Targeted cargo protein combination therapy

ABSTRACT

The present invention combines a targeted cargo protein with an active agent for the treatment of a disease or condition.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/105,408 filed Oct. 14, 2008. The contents of that application ishereby incorporated by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was created in the performance of a Cooperative Researchand Development Agreement with the Food and Drug Administration, anAgency of the Department of Health and Human Services. The Government ofthe United States has certain rights in this invention.

FIELD

This invention relates to compositions and methods for treating diseasesand conditions using targeted cargo proteins in combination with anotheractive agent.

BACKGROUND

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal humanmalignancies (32,000 deaths per year). Because of its aggressive growthand rapid metastasis to lymph nodes and liver, only 10% to 15% ofpatients are found to be resectable at diagnosis (1). Currently, themost common strategy for the treatment of advanced pancreatic cancer istreatment with gemcitabine, although the median survival time continuesto be <6 months for these patients (2, 3). Recently, several types ofinhibitors targeting the epidermal growth factor (EGF) receptor,platelet-derived growth factor (PDGF) receptor, and nuclear factor-nB(NF-nB) have shown their effectiveness in pancreatic cancer in murinemodels (4-7). In clinical trials, EGF receptor tyrosine kinase inhibitor(Erlotinib, Tarceva) plus gemcitabine enhances 1-year survival forpatients with advanced pancreatic cancer (8). However, the difference inmedian survival between Erlotinib plus gemcitabine group and gemcitabinealone group is <1 month. An effective new approach is needed formanagement of patients with this disease.

Gemcitabine (Gemzar) is a widely accepted first-line therapy foradvanced pancreatic cancer, although the median survival time continuesto be <6 months for these patients (2, 3). As most studies using singleagent show low response rate and little effect on patient survival inadvanced adenocarcinoma, several clinical trials using a combinedapproach of radiotherapy and/or molecular target therapy withgemcitabine have been initiated (33). In vitro studies have reportedsynergistic effect of gemcitabine with cisplatin, fluvastatinhydroxymethylglutaryl-CoA reductase inhibitor,CpG-oligodeoxynucleotides, EGFR, PDGF, and vascular endothelial growthfactor inhibitor targeting drugs (6, 34-37). In addition, immunotoxinswere shown to exert synergistic effect with chemotherapeutic drugs, forexample, doxorubicin plus anti-B4-blocked ricin, Ara-C plus granulocytemacrophage colony-stimulating factor fused to truncated diphtheria toxin(DT388-GM-CSF), and fludarabine with rituximab saporin-S6 conjugatedprotein (38-40).

SUMMARY

The disclosure describes proteins and other moieties that interact orbind to target cells such as cancer cells using a targeting moiety thatis linked to a protein or other toxic agent that kills or inhibitsgrowth or function of the target cells. The protein or other toxic agentthat kills or inhibits cancer cell growth is referred to as a cargomoiety and the cargo moiety linked to the targeting moiety iscollectively referred to as a targeted cargo protein. As describedherein, these targeted cargo proteins are used in combination withactive agents know to be effective in treating cancer to synergisticallyenhance the treatment of cancer in a mammalian subject, such as a human.The active agents used in combination with the targeted cargo proteinsmay be chemotherapeutic agents, antibodies or other agents typicallyused to treat cancer or other diseases or conditions. Targeting cellsurface receptors with targeted cargo proteins provides a uniqueopportunity for tumor therapy.

The invention is based in part on the unexpected discovery that atargeted cargo protein targeted against the IL-4 receptor, interleukin-4(IL-4) cytotoxin (an embodiment of which is also known as PRX321), whencombined with gemcitabine, a chemotherapeutic agent currently used totreat advanced pancreatic cancer, is shown to have a synergisticanti-tumor effect both in vitro and in a clinically relevant mouse modelof advanced pancreatic cancer. Specifically, those mice treated with acombination of PRX321 and gemcitabine showed a significant decrease intumor burden and improved survival compared to treatment with eitherPRX321 or gemcitabine alone. This study demonstrates for the first timethe potential of combining an IL-4 cytotoxin such as PRX321 with achemotherapeutic agent for treating patients with pancreatic cancer. Thedevices and methods of the present invention are directed to alleviatingthe above-described problems with previous treatments and, in addition,provide improved therapeutic results in comparison to IL-4-cytotoxin(IL4-PE) alone. It is believed that unique target expression on PDA andsynergistic effect of two drugs having independent mechanisms of actioncontribute to the overall improved therapeutic results.

Here, we show the efficacy of the combination therapy of gemcitabinewith PRX321 in animal models of pancreatic ductal adenocarcinoma (PDA).Targeting cell surface receptor with targeted cargo proteins (e.g.cytotoxins or immunotoxins) provides a unique opportunity for tumortherapy. Targeted cargo proteins offer the advantage of enhancedspecificity and direct toxicity for tumor cells that over-express thereceptor, thus limiting the potential toxicity to normal tissues (9).Several clinical trials using PRX321, IL-13 cytotoxin, and recombinantimmunotoxin BL22 have shown survival benefits in patients withglioblastoma multiforme, chronic lymphocytic leukemia, and hairy cellleukemia (10-13).

Interleukin-4 (IL-4) is an important Th2-derived cytokine, which isinvolved in mediating antitumor immune-modulating activities (14). IL-4has been shown to have a modest but direct inhibitory effect on thegrowth of several tumor cells in vitro and in vivo (15, 16). Based onthese properties, IL-4 was tested in the clinic as a treatment forhematopoietic and solid malignancies, but it showed limited antitumoractivity (17). To improve this limited activity, we targeted IL-4receptor (IL-4R) because a variety of human tumor cells, includingpancreatic cancer, express high-affinity receptors for IL-4 (18-22).

We show herein that 60% of PDA samples express moderate- to high densitysurface IL-4Rs, whereas normal pancreas express no or very low levels ofIL-4R. PRX321 is highly cytotoxic to pancreatic cancer cell lines;however, it was not cytotoxic to HPDE cells, fibroblasts, and HUVEC,which express no or low levels of IL-4R. We also show that PRX321synergizes with gemcitabine in mediating cytotoxic activity inpancreatic cancer cell lines in vitro, and in animal models of humanpancreatic cancer in vivo. A significant prolonged survival effect ofPRX321 and its combination with gemcitabine was shown in mice with earlydisease. Forty percent of mice that received combination therapy showedcomplete eradication of pancreatic tumors. In addition, this significantsurvival benefit was also confirmed in animals implanted with theclinical pancreatic cancer sample.

The foregoing and other features of the disclosure will become moreapparent from the following detailed description, which proceeds withreference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of the structure and amino acidsequence of an exemplary targeted cargo protein, a circularly permutedIL-4-Pseudomonas toxin, PRX321 (SEQ ID NO: 1). Disulfide bonds areindicated on the drawing.

FIG. 2A shows the visible fluorescent area quantification of tumor sizeas a function of time (obtained from sequential whole-body imaging) inan early pancreatic tumor model in which tumor bearing mice received notreatment, treatment with gemcitabine alone, treatment with IL-4cytotoxin (PRX321) alone, or a combination of gemcitabine and PRX321.

FIG. 2B shows Kaplan-Meier survival curves in an early pancreatic tumormodel.

FIG. 3A shows the visible fluorescent area quantification of tumor sizeas a function of time (obtained from sequential whole-body imaging) inan advanced pancreatic tumor model in which tumor bearing mice receivedno treatment, treatment with gemcitabine alone, treatment with IL-4cytotoxin (PRX321) alone, or a combination of gemcitabine and PRX321.

FIG. 3B shows Kaplan-Meier survival curves in an advanced pancreatictumor model.

FIG. 4 shows Kaplan-Meyer survival curves in an advanced pancreaticcancer model generated from a clinical sample by orthotopictransplantation in SCID mice.

DETAILED DESCRIPTION

While the invention has been described in some detail by way ofillustration and example, it should be understood that the invention issusceptible to various modifications and alternative forms, and is notrestricted to the specific embodiments set forth in the Examples. Itshould be understood that these specific embodiments are not intended tolimit the invention but, on the contrary, the intention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

The present invention involves treating a disease or condition in asubject by administering to the subject at least one targeted cargoprotein of the present invention in combination with an active agentknown to be effective in treating the disease or condition.

In some embodiments of the invention, the targeted cargo proteincomprises a toxin, and is thus a cytotoxin or immunotoxin. Preferably,the targeted cargo protein is a cytotoxin or immunotoxin that bindsspecifically to the IL-4 receptor, for example, an IL-4-cytotoxin suchas PRX321.

In accordance with the present invention, the active agent may be anysubstance or the like, or treatment protocol or the like, that providesa therapeutic benefit to the patient when combined with theadministration of a targeted cargo protein. Active agents include, butare not limited to chemotherapeutic agents. It is intended that anactive agent may be a substance that uses a different mechanism ofaction than the targeted cargo protein, or it may be a substance thatuses the same mechanism of action.

In some embodiments of the invention, the active agent is achemotherapeutic agent. In most preferred embodiments of the invention,the active agent is gemcitabine or doxarubicin.

In some embodiments of the invention, the disease or condition is anydisease or condition characterized by cells having a unique oridentifying expression pattern of a surface molecule or target. In someembodiments of the invention, the cell surface molecule is a receptor.In some preferred embodiments of the invention, the receptor is an IL-4receptor (IL-4 R).

Although the IL-4 R is expressed at low levels by certain normal cells,such as resting T lymphocytes, B lymphocytes and resting or activated CD34 bone marrow cells, it is over-expressed in a wide range of solidtumors, including, for example, brain cancer, including malignantastrocytoma and gliobastoma multiforme, Kaposi sarcoma, bladder cancer,renal cell cancer, breast cancer, pancreatic cancer, non-small cell lungcancer, thyroid cancer, squamous cell carcinoma of the head and neck,colon cancer and other cancers of the gastrointestinal system,mesothelioma and prostate cancer. As used herein, when a cell surfacemolecule is over-expressed or uniquely expressed in cells characterizinga disease or condition such as cancer, a targeted cargo protein thatbinds specifically to the cell surface molecule is said to be specificfor the cell associated with the disease or condition.

In some embodiments of the invention, the disease or condition is acancer or tumor producing disease. In some embodiments of the invention,the cancer is a pancreatic cancer. In preferred embodiments of theinvention, the pancreatic cancer is pancreatic ductal adenocarcinoma(PDA). In other preferred embodiments of the invention, the disease orcondition is any disease or condition that over-expresses IL-4receptors. Conditions, as used herein, include, but are not limited toinflammation.

In some embodiments, the invention provides methods and compositions forinhibiting the growth of a target cell, said target cell comprising acell characterized by having over-expression of an IL-4 receptor. Theinvention includes contacting the cell with a targeted cargo protein,said targeted cargo protein comprising a targeting moiety and a proteinsynthesis-inhibiting moiety, and then, within a pre-determined andmedically appropriate time period (e.g., one week), at least one activeagent. In some embodiments of the invention, the pre-determined timeperiod may be selected from the time periods consisting of within 96hours, within 72 hours, within 48 hours, and within 24 hours. In someembodiments, the cell is concurrently contacted with both the targetedcargo protein and the first active agent.

The present invention also includes a method of treating a disease orcondition by administering a targeted cargo protein in combination withan active agent.

As used herein, “in combination” or variations on that phrase, refer toadministering the targeted cargo protein and the active agent within aclose enough time period that the patient derives a beneficial resultthat would not have occurred if the targeted cargo protein and theactive agent were not administered in combination. In some embodimentsof the invention, in combination refers to administering the targetedcargo protein and the active agent in the same composition; in otherembodiments the targeted cargo protein and the active agent areadministered sequentially or serially. In some embodiments of theinvention, the targeted cargo protein and the active agent areadministered are part of an integrated therapeutic plan. In some ofthese exemplary embodiments, the targeted cargo protein and the activeagent may be administered hours or days, weeks, or months apart, but thecombination of the two agents provides a beneficial result for thepatient.

The composition of the present invention may also be administeredrepetitively. For example, a cargo protein may administered alone or incombination with an active agent, then the cargo protein may beadministered again, alone or in combination with an active agent, at anytherapeutically appropriate interval (e.g., the next day or after aweek).

One skilled in the art will recognize that the invention as describedhere may be reconfigured into different combinations, elements, andprocesses which are included within the scope of the invention.

It is possible to deliver IL4-PE through drug leaching stents forlocalized cancer to avoid systemic exposure and stent serve as a“reservoir”

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular forms“a,” “an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. For example, the term “comprising a targetedcargo protein” includes single or plural targeted cargo proteins and isconsidered equivalent to the phrase “comprising at least about onetargeted cargo protein.” The term “or” refers to a single element ofstated alternative elements or a combination of two or more elements,unless the context clearly indicates otherwise. As used herein,“comprises” means “includes.” Thus, “comprising A or B,” means“including A, B, or A and B,” without excluding additional elements.

(A) Active agent, as used herein, refers to any substance that providesa therapeutic benefit to the patient. Active agents include but is notlimited to chemotherapeutic agents; anti-disease drugs; anti-diseasechemicals, immune system mediators, including enhancers and inhibitors;immune suppressive compounds such as cyclosporine and retinoic acidwhich has shown to enhance the cytotoxic activity of ricin A. Othertherapeutic agents include toxins which are not required to beinternalized into the target cell such as PRX302 which acts on the cellsurface by creating pores.

Exemplary active agents include but are not limited to those disclosedin PCT/US2008/002747 (Pastan, et al.), incorporated herein by reference.The preferred active agents are gemcitabine and doxorubicin. Someadditional exemplary active agents are protein synthesis inhibitors suchas L-asparaginase, cyctotoxic/antitumor antibiotics, such asdaunorubicin, dosorubicin epirubicin, idarubicin, mitoxantrone,valrubicin, bleomycin, hyroxyurea, and mitomycin. Other active agentsinclude plant alkaloids, such as docetaxel, paclitaxel, vinblastine,vincristine, vindesine, and vinorelbine. Other active agents includealkylating agents, such as nitrogen mustards (such as chlorambucil,chlormethine, cyclophosphamide, ifosfamide or melphalan), a nitrosoureas(such as carmustine, lomustine, spreptozocin), platinum drugs (such ascarboplatin, cisplatin, oxaliplatin, BBR3464), busulfan, dacarbazine,mechlorethamne, procarbazine, temozolomide, thioTEPA, uramustine),antimetabolites (such as aminopterin, methotrexate, pemetrexed,ralititrexed, cladribine, clofarabine, fludarabine, mercaptopurine,thioguanine, pentostatin, capecitabine, cytarabine, fluorouracil andgemcitabine). Other active also include topoisomerase inhibitors such astopotecan, irinotecan, podophyllum, etoposide and teniposide. Activeagents also include therapeutic antibodies (such as alemtuzumab,bevacizumab, cetuximab, gentuzumab, pantitumumab, rituximab, tositmomab)and kinase inhibitors (such as imatinib, nilotinib, dasatinib,erlotinib, gefitinib, lapatinib, sorafenib, sunitinib and vandetanib).

Combinations of active agents may be used in the methods of theinvention. For example the FOLFOX protocol, which involvesadministration of three agents, folinic acid, fluorouracil andoxaliplatin, may be used in combination with one or more targeted cargoproteins.

Adjuvants or immune-stimulants such as BCG (Bacillus Calmette Geurin)may also be used in combination with targeted cargo proteins.

(B) “Over-expresses” or similar terms, refers to the presence of areceptor, or the presence of a receptor in an amount significantlyhigher than normal. Typically, such over-expression is an indicator of adisease or condition. 11-4 receptor is one known example. EGFR and VEGFRreceptors may also be targeted, in accordance with the presentinvention.

(C) Targeted cargo protein consists of a targeting moiety linked to acargo moiety. In accordance with the present invention, a targetingmoiety is any compound that binds to a molecule (herein referred to as atarget) displayed on the target cell surface. A targeting moiety can bean antibody that binds to a target (e.g. receptor), a ligand (e.g.,cytokine or growth factor) that bind to a receptor, a permuted ligandthat binds to a receptor or a peptide sequence sensitive to cleavage bya tumor-associated protease. Exemplary targeting moieties include butare not limited to those disclosed in U.S. Pat. No. 6,011,002,incorporated herein by reference. Some exemplary targeting moieties andexemplary GenBank accession numbers are shown in Table 1 below.Typically, targeting moieties selectively bind to one type of celldisplaying a target more effectively than they bind to other types ofcell that do not display the target, or that display the target at lowlevels.

TABLE 1 Exemplary targeting moiety sequences Receptor or Antigen to beTargeted Accession Number* Epidermal growth factor (EGF) NP_001954;EAX06257.1; AAR84237.1 Vascular endothelial growth AAA35789; CAC19515factor (VEGF) Interleukin 2 (IL-2) CAA07317; AAB46883.1; NP_000577.2Interleukin 3 (IL-3) AAC08706.1; AAA99502.1; CAE45598.1 Interleukin 4(IL-4) AAH70123; CAA57444.1; AAH67515.1 (also see SEQ ID NO: 2 andvarious circularly permuted ligands in U.S. Pat. No. 6,011,002) IL-5NP_000870.1; CAA01794.1; P32927.2 IL-13 AAH96141.2; AAH96138.1;AAH96139.1 Granuclocyte-macrophage colony P04141.1; AAI13925.1;AAI08725.1 stimulating factor (GMCSF) Granulocyte colony stimulatingQ99062; P09919 factor (GCSF) Tenascin AAA36728.1; CAA39628.1;NP_002151.2 Mesothelin CAC37289.1; ABW03459.1; AAH09272.1; AAH03512.1;as well as the mesothelins disclosed in U.S. Pat. Nos. 7,081,518 and6,051,405 (mesothelin sequences therein herein incorporated byreference) CD22 BAA36575.1; BAA36576.1; BAA36567.1 PSMA (also known asfolate ABO93402.2; AAC83972.1; NP_001014986.1; hydrolase) NP_004467.1*GenBank Numbers are herein incorporated by reference, as well as theircorresponding nucleic acid sequences.

The cargo moiety may be derived from plant, animal, or bacteria. Inaccordance with the present invention, cargo moieties function tosignificantly kill, reduce or inhibit the growth of target cells. Acargo moiety may be a peptide (e.g. protein fragment or full lengthprotein) or other molecule that can function to significantly reduce orinhibit a target cell. In some examples, the cargo moiety is not apeptide, but another molecule that can function to significantly reduceor inhibit the growth of target cells, such as thapsigargin. Exemplarycargo moieties include cytotoxins, such as Pseudomonas exotoxin (PE),diphtheria toxin (DT), including but are not limited to those disclosedin PCT/US2008/002747 (Pastan, et al.), incorporated herein by reference.In other examples, cargo moieties are proteins that normally contributeto the control of cell life cycles, for example a cargo protein cantrigger cell death, such as via apoptotic pathways (e.g. Bad, Bax andother pro-apoptotic members of the Bcl-2 family of proteins). Someexemplary cargo moieties and exemplary GenBank accession numbers areprovided in Table 2 below.

TABLE 2 Exemplary cargo moiety sequences Cargo Moiety Accession NumbersDiphtheria ABU25232; CAA24778 toxin (DT) Aerolysin ABR14715.1;ABR14714.1 Proaerolysin AAA21938.1; P09167.2; U.S. Pat. No. 7,282,476(proaerolysin sequences therein herein incorporated by reference)Bouganin AAL35962 and SEQ ID NO: 9 in U.S. Pat. No. 6.737,511, as wellas variant sequences provided in U.S. Pat. No. 7,339,031 and WO2005/090579 (bouganin sequences therein herein incorporated byreference) Pseudomonas 1IKP A; AAB59097.1; AAF90003.1 (also see SEQ IDNO: 1 of U.S. Pat. No. exotoxin (PE) 6,011,002) Bcl-2 pro- BAD:CAG46757; AAH01901.1; CAG46733.1; and sequences provided in U.S.apoptotic Pat. No. 6,737,511 proteins such BAX: CAE52909.1; AAO22992.1;EAW52418.1 as BAD and BAX Cholera toxin BAA06291.1; ACF35010.1;BAA06288.1; as well as variant sequences provided in US patentapplication No. 61/058,872 (variant cholera toxin sequences thereinherein incorporated by reference) Ribonuclease A BAA05124.1;NP_937877.1; NP_115961.2; Q5GAN4.1; and sequences provided in PCTPublication No. WO 2007/041361 (rapLR1 sequences therein hereinincorporated by reference) *GenBank Numbers are herein incorporated byreference, as well as their corresponding nucleic acid sequences.

In addition to native targeting moieties and cargo moieties, variantsequences can also be used, such as mutant sequences with greaterbiological activity than that of the native sequence.

The preferred targeted cargo proteins are IL-4-PE, IL-4-BAD, IL-4-DT andIL-4-doxarubicin.

(D) Inhibiting or inhibition or similar terms refers to cell killing,cell inhibition, loss of cell function, or any other action, direct orindirect, that results in or mediates cell viability and/or function. Inpreferred embodiments, the compositions of the invention include atleast two different mechanisms of action, e.g., two mechanisms of cellkilling (such as apoptosis and necrosis).

(E) Contacting refers to placement in direct physical association. Withrespect to therapeutic targeted cargo proteins and active agents, suchtherapeutics are considered to contact a target cancer cell, such as apancreatic cancer cell, in a subject if the therapeutics areadministered to the subject by a route that is generally accepted in theart for administering that type of therapeutic.

(F) Specific or specific binding refers to a preferential bindingbetween an agent and a specific target. For example, specific bindingrefers to the situation when a targeted cargo protein that includes atargeting moiety specific for a molecule or receptor displayed on acancer cell binds to the cancer cell but does not significantly bind toother cells that do not display the target but are in close proximity tothe cancer cell. Specific binding interactions are mediated by one or,typically, more noncovalent bonds between the binding molecules. Incontrast to non-specific binding sites, specific binding sites aresaturable. One exemplary way to characterize specific binding is by aspecific binding curve. A specific binding curve shows, for example, theamount of one binding partner (the first binding partner) bound to afixed amount of the other binding partner as a function of the firstbinding partner concentration. As the first binding partnerconcentration increases under these conditions, the amount of the firstbinding partner bound will saturate. In another contrast to non-specificbinding sites specific binding partners involved in a direct associationwith each other (e.g., a protein-protein interaction) can becompetitively removed (or displaced) from such association (e.g.,protein complex) by excess amounts of either specific binding partner.Such competition assays are very well known in the art. If a targetedcargo protein exhibits specific binding to a cell surface molecule on acancer cell, it is said to be specific for its target on the cancercell.

(G) Antibody or antibodies refer to Immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, that is,molecules that contain an antigen binding site that specifically binds(immunoreacts with) an epitope, such as an epitope displayed by cancercells or another target cell. Antibodies include monoclonal antibodies,polyclonal antibodies, as well as humanized antibodies. Antibodies alsoinclude affibodies. Affibodies mimic monoclonal antibodies in functionbut are based on Protein A. Affibodies can be engineered ashigh-affinity ligands for binding to a targeting moiety.

(H) Subject refers to a living multi-cellular vertebrate organisms,including human a non-human mammal.

Description of exemplary embodiments of targeting moieties and cargomoieties and their combinations that are useful in the methods of theinvention are the following:

Targeting Moieties

In addition to the targeting moieties described above, it will beappreciated that targeting moieties (as well as protein-based cargomoieties) may be truncated or modified and still have the same or evenmore biological activity. Therefore, the invention includes variants oftargeting moieties and cargo moieties and portions, fragments orsubunits thereof that have at least 60% sequence identity, at least 75%,at least 80%, at least 85%, at least 90%, at least 98%, or even at least99% sequence identity to the native protein sequences or fragments fromwhich they are derived, as long as the variants retain, or haveenhanced, desired biological activity. In some examples, variantsequences retain substantially the same amount of even more of thenative biological function of the parent protein, such as the ability toactivate an intracellular signal cascade. However, useful varianttargeting moiety molecules may in some examples retain little or nobiological activity, but retain the ability to bind the appropriatetarget with high specificity, and such molecules are included within thescope of the invention.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation can be found at the NCBI web site.

BLASTN can be used to compare nucleic acid sequences, while BLASTP canbe used to compare amino acid sequences. To compare two nucleic acidsequences, the options can be set as follows: -i is set to a filecontaining the first nucleic acid sequence to be compared (such asC:\seq1.txt); -j is set to a file containing the second nucleic acidsequence to be compared (such as C:\seq2.txt); -p is set to blastn; -ois set to any desired file name (such as C:\output.txt); -q is set to−1; -r is set to 2; and all other options are left at their defaultsetting. For example, the following command can be used to generate anoutput file containing a comparison between two sequences: C:\Bl2seqc:\seq1.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q −1 -r 2.

To compare two amino acid sequences, the options of Bl2seq can be set asfollows: -i is set to a file containing the first amino acid sequence tobe compared (such as C:\seq1.txt); -j is set to a file containing thesecond amino acid sequence to be compared (such as C:\seq2.txt); -p isset to blastp; -o is set to any desired file name (such asC:\output.txt); and all other options are left at their default setting.For example, the following command can be used to generate an outputfile containing a comparison between two amino acid sequences: C:\B12seq c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If thetwo compared sequences share homology, then the designated output filewill present those regions of homology as aligned sequences. If the twocompared sequences do not share homology, then the designated outputfile will not present aligned sequences.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a nucleic acid sequence that has1166 matches when aligned with a test sequence having 1554 nucleotidesis 75.0 percent identical to the test sequence (1166÷1554*100=75.0). Thepercent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer.

For comparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). Homologs are typically characterizedby possession of at least 70% sequence identity counted over thefull-length alignment with an amino acid sequence using the NCBI BasicBlast 2.0, gapped blastp with databases such as the nr or swissprotdatabase. Queries searched with the blastn program are filtered withDUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70).Other programs use SEG. In addition, a manual alignment can beperformed. Proteins with even greater similarity will show increasingpercentage identities when assessed by this method, such as at leastabout 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a cargoprotein or targeting moiety provided herein.

When aligning short peptides (fewer than around 30 amino acids), thealignment is performed using the Blast 2 sequences function, employingthe PAM30 matrix set to default parameters (open gap 9, extension gap 1penalties). Proteins with even greater similarity to the referencesequence will show increasing percentage identities when assessed bythis method, such as at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99% sequence identity to a cargo moiety or targeting moietyprovided herein. When less than the entire sequence is being comparedfor sequence identity, homologs will typically possess at least 75%sequence identity over short windows of 10-20 amino acids, and canpossess sequence identities of at least 85%, 90%, 95% or 98% dependingon their identity to the reference sequence. Methods for determiningsequence identity over such short windows are described at the NCBI website.

Antibodies or fragments thereof may be used as targeting moieties. Anaturally occurring antibody (e.g., IgG, IgM, IgD) includes fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinterconnected by disulfide bonds. However, it has been shown that theantigen-binding function of an antibody can be performed by fragments ofa naturally occurring antibody. Thus, these antigen-binding fragmentsare also intended to be designated by the term “antibody.” Specific,non-limiting examples of binding fragments encompassed within the termantibody include (i) a Fab fragment consisting of the VL, VH, CL and CH1domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii)an Fv fragment consisting of the VL and VH domains of a single arm of anantibody (scFv) and scFv molecules linked to each other to form abivalent dimer (diabody) or trivalent trimer (triabody); (iv) a dAbfragment (Ward et al., Nature 341:544-546, 1989) which consists of a VHdomain; (v) an isolated complimentarily determining region (CDR); and(vi) a F(ab′)2 fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region.

Methods of producing polyclonal and monoclonal antibodies are known tothose of ordinary skill in the art, and many antibodies are available.See, e.g., Coligan, Current Protocols in Immunology Wiley/Greene, N Y,1991; and Harlow and Lane, Antibodies: A Laboratory Manual Cold SpringHarbor Press, N Y, 1989; Stites et al., (eds.) Basic and ClinicalImmunology (4th ed.) Lange Medical Publications, Los Altos, Calif., andreferences cited therein; Goding, Monoclonal Antibodies: Principles andPractice (2d ed.) Academic Press, New York, N.Y., 1986; and Kohler andMilstein, Nature 256: 495-497, 1975. Other suitable techniques forantibody preparation include selection of libraries of recombinantantibodies in phage or similar vectors. See, Huse et al., Science 246:1275-1281, 1989; and Ward et al., Nature 341: 544-546, 1989.

Immunoglobulins and certain variants thereof are known and many havebeen prepared in recombinant cell culture (e.g., see U.S. Pat. No.4,745,055; U.S. Pat. No. 4,444,487; WO 88/03565; EP 256,654; EP 120,694;EP 125,023; Faoulkner et al., Nature 298:286, 1982; Morrison, J.Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol 2:239, 1984).Detailed methods for preparation of chimeric (humanized) antibodies canbe found in U.S. Pat. No. 5,482,856. Additional details on humanizationand other antibody production and engineering techniques can be found inBorrebaeck (ed), Antibody Engineering, 2nd Edition Freeman and Company,N Y, 1995; McCafferty et al., Antibody Engineering, A PracticalApproach, IRL at Oxford Press, Oxford, England, 1996, and Paul AntibodyEngineering Protocols Humana Press, Towata, N.J., 1995.

In some examples, an antibody specifically binds to a target protein(e.g., a cell surface receptor such as an IL4 receptor) with a bindingconstant that is at least 10³ M⁻¹ greater, 10⁴ M⁻¹ greater or 10⁵ M⁻¹greater than a binding constant for other molecules in a sample. In someexamples, a specific binding reagent (such as an antibody (e.g.,monoclonal antibody) or fragments thereof) has an equilibrium constant(K_(d)) of 1 nM or less. For example, a specific binding agent may bindto a target protein with a binding affinity of at least about 0.1×10⁻⁸M, at least about 0.3×10⁻⁸M, at least about 0.5×10⁻⁸ M, at least about0.75×10⁻⁸ M, at least about 1.0×10⁻⁸M, at least about 1.3×10⁻⁸ M atleast about 1.5×10⁻⁸M, or at least about 2.0×10⁻⁸ M. Kd values can, forexample, be determined by competitive ELISA (enzyme-linked immunosorbentassay) or using a surface-plasmon resonance device such as the BiacoreT100, which is available from Biacore, Inc., Piscataway, N.J.

IL-4 is a pleiotropic cytokine produced by activated T cells, and is theligand for the IL-4 receptor. The IL-4 receptor also binds to IL-13.Thus, IL-13 can also be used as a targeting moiety to target the IL-4receptor. IL-4, IL-3, IL-5, IL-13, and CSF2 form a cytokine gene clusteron human chromosome 5q, with this gene particularly close to IL-13.Exemplary IL-4 and IL-13 proteins that can be used in the targeted cargoproteins of the present disclosure include those provided in Table 1, aswell as sequences having at least 60% sequence identity, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98% oreven at least 99% sequence identity to such sequences, as long as thevariant retains the ability to bind the IL-4 receptor.

IL-4 (including IL-4 circularly permuted ligands and other IL-4 receptorbinding proteins such as IL-13) are targeting moieties that can belinked to BCL-2 family proteins, such as BAX, BAD, BAT, BAK, BIK, BOK,BID BIM, BMF and BOK, or a toxin such as aerolysin, proaerolysin,Pseudomonas exotoxin, or combinations thereof. Any form or derivative ofIL-4 can be used as the targeting moiety. For example, IL-4 or fragmentsof IL-4 that bind to the IL-4 receptor can be used. Additionally,multiple cargo moieties can be linked to IL-4 or multiple IL-4 proteinscan be linked to cargo moieties.

Antibodies (including fragments, humanized antibodies and the like asdescribed above) that specifically bind to IL-4 receptors can be linkedto BCL-2 family proteins, such as BAX, BAD, BAT, BAK, BIK, BOK, BID BIM,BMF and BOK, or a toxin such as aerolysin, proaerolysin, Pseudomonasexotoxin, or combinations thereof. Antibodies are commercially availablefrom various companies such as Millipore, Bedford, Mass. or custom madeantibodies can be ordered from companies such as Cambridge ResearchBiochemicals.

Cargo Moieties

Cargo moieties that are useful in the methods of the invention reduce orinhibit target cells. As described above, some examples of cargomoieties are not proteins, but other molecules, such as chemotherapeuticagents. For example, toxins and proteins that function to control celllife cycles can be used as cargo moieties. Toxins that can be used ascargo moieties include toxins made by microorganisms, plants or animals,as well as toxins made by human cells. Similarly, any natural cellgrowth controlling protein can be used as a cargo moiety. For example,proteins that trigger cell death during the normal life cycle of anorganism can be used as cargo moieties. In some examples, an oncolyticvirus (e.g., see Allen et al., Mol. Ther. 16:1556-64, 2008) or liposomescarrying cytotoxic agents (e.g., see Madhankumar et al., Mol. Cancer.Ther. 5:3162-9, 2006) is used as the cargo protein.

In one example, the cargo moiety is a toxin. Toxins that are cytotoxicmay be herein referred to as “cytotoxins.” Exemplary toxins that can beused include pore-forming toxins, and toxins that upon internalizationinhibit cell growth. In other examples, cargo moieties are proteins thatare apoptotic triggering proteins, and cell growth inhibiting proteins.In some examples, the toxin is a modified bacterial toxin such that theresulting toxin is less immunogenic than the native toxin. Such modifiedtoxins, such as a modified Pseudomonas exotoxin A, can reduce thepatient's immunogenic response, thereby allowing repeatedadministration.

Pore forming toxins are toxins that form pores in the cell membranethereby killing the cell via cell lyses. Exemplary pore forming toxinsinclude but are not limited to human toxins such as perforin orbacterial toxins such as aerolysin as well as modified pore-formingprotein toxins that are derived from naturally occurring pore-formingprotein toxins (nPPTs) such as aerolysin or aerolysin-relatedpolypeptides. Suitable aerolysin-related nPPTs have the followingfeatures: a pore-forming activity that is activated by removal of aninhibitory domain via protease cleavage, and the ability to bind toreceptors that are present on cell membranes through one or more bindingdomains. In some examples the linker can be engineered to be sensitiveto a protease or be chemically liable. Additional examples of poreforming toxins that can be used as cargo moieties include, but are notlimited to, proaerolysin from Aeromonas hydrophila, Aeromonas trota andAeromonas salmonicida, alpha toxin from Clostridium septicum, anthraxprotective antigen, Vibrio cholerae VCC toxin, epsilon toxin fromClostridium perfringens, and Bacillus thuringiensis delta toxins. Adetailed description of the engineering of proaerolysin can be found inU.S. Pat. No. 7,282,476, which is herein incorporated by reference.

Additional toxins that can be used as cargo moieties include toxins thatact within a cell. For example, anthrax, diphtheria, cholera, andbotulinum toxins include a portion that acts in the cytoplasm, as wellas a portion that acts to bind to the cell surface. These toxins, orportions thereof, can be linked to a targeting moiety and used toinhibit cancer cancer cell growth. Select members of the ribonuclease A(RNase A) superfamily are potent cytotoxins. These cytotoxicribonucleases enter the cytosol, where they degrade cellular RNA andcause cell death.

In some examples ribosome inactivating proteins can be used as toxins.In these examples the cargo moiety is a polypeptide havingribosome-inactivating activity including, without limitation, gelonin,bouganin, saporin, ricin, ricin A chain, bryodin, restrictocin, andvariants thereof. Diphtheria toxin and Pseudomonas exotoxin A inhibitprotein synthesis via ADP-ribosylation of elongation factor 2. When thecargo moiety is a ribosome-inactivating protein or inhibits proteinsynthesis via ADP-ribosylation of elongation factor 2, the targetedcargo protein can be internalized upon binding to the cancer cell.

Cargo moieties that induce apoptosis can also be used to target cancercells. Examples of cargo moieties that induce apoptosis includecaspases, granzymes and BCL-2 pro-apoptotic related proteins such as BAX(e.g., Accession no: CAE52910), BAD (e.g., Accession no: CAG46757), BAT(e.g., Accession no: AAI07425), BAK (e.g., Accession no: AAA74466), BIK(e.g., Accession no: CAG30276), BOK (e.g., Accession no: AAH06203), BID(e.g., Accession no: CAG28531), BIM (e.g., Accession no: NP 619527) andBMF (e.g., Accession no: AAH69328). These cargo moieties can be usedalone or in combination to reduce or inhibit cancer cell growth.

Aerolysin is a channel-forming toxin produced as an inactive protoxincalled proaerolysin (PA). Exemplary aerolysin and PA sequences that canbe used in a targeted cargo protein are provided in Table 2. The PAprotein contains many discrete functionalities that include a bindingdomain, a toxin domain, and a C-terminal inhibitory peptide domain thatcontains a protease activation site. The binding domain recognizes andbinds to glycophosphatidylinositol (GPI) membrane anchors, such as arefound in Thy-1 on T lymphocytes, the PIGA gene product found inerythrocyte membranes and Prostate Stem Cell Antigen (PSCA). Theactivation or proteolysis site within proaerolysin is a six amino acidsequence that is recognized as a proteolytic substrate by the furinfamily of proteases. PA is activated upon hydrolysis of a C-terminalinhibitory segment by furin. Activated aerolysin binds to GPI-anchoredproteins in the cell membrane and forms a heptamer that inserts into themembrane producing well-defined channels of ˜17 Å. Channel formationleads to rapid cell death. Wild-type aerolysin is toxic to mammaliancells, including erythrocytes, for example at 1 nanomolar or less.

In some examples, a target cargo protein is a PA molecule with thenative furin site replaced with a different cleavage site, such asprostate-specific protease cleavage site (e.g., a PSA-specific cleavagesite, which permits activation of the variant PA in the presence of aprostate-specific protease such as PSA, PMSA, or HK2). In one example, aprostate-specific protease cleavage site is inserted into the nativefurin cleavage site of PA, such that PA is activated in the presence ofa prostate-specific protease, but not furin. In another example, avariant PA molecule further includes a functionally deleted bindingdomain (e.g., about amino acids 1-83 of a native PA protein sequence).Functional deletions can be made using any method known in the art, suchas deletions, insertions, mutations, or substitutions. In some examples,targeted cargo proteins include variant PA molecules in which the nativebinding domain is functionally deleted and replaced with aprostate-tissue or other tissue-specific binding domain. In otherexamples, variant PA molecules include a furin cleavage site and afunctionally deleted binding domain which is replaced with aprostate-tissue specific binding domain. Such variant PA molecules aretargeted to prostate cells via the prostate-tissue specific bindingdomain, and activated in the presence of furin.

Bouganin is a ribosome-binding protein originally isolated fromBougainvillea speotabilis (see U.S. Pat. No. 6,680,296). Exemplarymodified bouganins are described in WO 2005/090579 and U.S. Pat. No.7,339,031. Bouganin damages ribosomes and leads to a cessation ofprotein synthesis and cell death. Exemplary bouganin proteins that canbe used in the targeted cargo proteins of the present disclosure includethose in GenBank Accession No. AAL35962, as well as those native andmodified bouganin sequences provided in U.S. Pat. Nos. 6,680,296;7,339,031 and PCT publication WO 2005/090579 (bouganin sequences hereinincorporated by reference), as well as sequences having at least 60%sequence identity, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98% or even at least 99% sequence identityto such sequences.

BAD, BCL2-associated agonist of cell death, is a regulator of programmedcell death (apoptosis). BAD positively regulates cell apoptosis byforming heterodimers with BCL-xL and BCL-2, and reversing their deathrepressor activity. Proapoptotic activity of BAD is regulated throughits phosphorylation. Exemplary BAD proteins that can be used in thetargeted cargo proteins of the present disclosure include those inGenBank Accession Nos. CAG46757; AAH01901.1; and CAG46733.1, as well asthose sequences provided in U.S. Pat. No. 6,737,511 (sequences hereinincorporated by reference), as well as sequences having at least 60%sequence identity, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98% or even at least 99% sequence identityto such sequences, as long as the variant retains or has enhancedbiological activity of the native BAD protein.

BAX, BCL2-associated X protein, is a regulator of programmed cell death(apoptosis). This protein forms a heterodimer with BCL2, and functionsas an apoptotic activator. BAX interacts with, and increases the openingof, the mitochondrial voltage-dependent anion channel (VDAC), whichleads to the loss in membrane potential and the release of cytochrome c.Exemplary BAX proteins that can be used in the targeted cargo proteinsof the present disclosure include those provided by GenBank AccessionNos. CAE52909.1; AAO22992.1; EAW52418.1, U.S. Pat. No. 6,645,490 (Bax inthe IL2-Bax construct is a Bax-alpha variant that can be used in thepresent disclosure), as well as sequences having at least 60% sequenceidentity, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98% or even at least 99% sequence identity to suchsequences, as long as the variant retains or has enhanced biologicalactivity of the native BAX protein.

In some examples, the BAX protein of a targeted cargo protein may bemodified such that the C-terminal anchor domain has been deleted andreplaced with a CaaX sequence. CaaX is a peptide with the sequenceCysteine-a-a-X where “X” is any amino acid and “a” is an aliphatic aminoacid. Because membrane association of BAX is needed for optimalapoptosis activity, addition of membrane binding domains such as CaaXcan enhance their pro-apoptotic activities. Proteins with CaaX sequenceare farnesylated. Farnesylated proteins are targeted to membranes (e.g.,see Wright and Philip, J. Lipid Res., 2006, 47(5): 883-91). PotentialBAX variants containing a CaaX sequence may or may not contain theC-terminal anchor domain.

Pseudomonas exotoxin (PE) is a toxin secreted by Pseudomonas. Native PEis cytotoxic tor mammalian cells due to its ability to enter cells byreceptor-mediated endocytosis and then, after a series of intracellularprocessing steps, translocate to the cell cytosol and ADP-ribosylateelongation factor 2. This results in the inhibition of protein synthesisand cell death. PE has three functional domains: an amino-terminalreceptor-binding domain, a middle translocation domain, and acarboxyl-terminal ADP-ribosylation domain. Modified PE molecules caninclude elimination of domain Ia, as well as deletions in domains II andIII. Exemplary PE proteins that can be used in the targeted cargoproteins of the present disclosure include those provided in Table 1, aswell as sequences having at least 60% sequence identity, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98% oreven at least 99% sequence identity to such sequences as long as thevariant retains or has enhanced biological activity of the native PEprotein.

Thapsigargin is an inhibitor of sarco/endoplasmic reticulum Ca2+ATPases. Thapsigargin is classified as a sesquiterpene lactone, andraises cytosolic calcium concentration by blocking the ability of thecell to pump calcium into the sarcoplasmic and endoplasmic reticulumwhich causes these stores to become depleted. Store-depletion cansecondarily activate plasma membrane calcium channels, allowing aninflux of calcium into the cytosol.

Ribonuclease A (RNAseA) is an endonuclease that cleaves single-strandedRNA. RNAse A toxins can be obtained from mammals and reptiles. ExemplaryRNAse A proteins that can be used in the targeted cargo proteins of thepresent disclosure include those provided in Table 2, as well assequences having at least 60% sequence identity, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98% or even atleast 99% sequence identity to such sequences, as long as the variantretains or has enhanced biological activity of the native RNAseA toxin.

The cargo moiety used can include native sequences (such as the GenBankAccession Nos. and sequences present in the patents referenced in Table2 and listed above), as well as variants thereof, such as a varianthaving at least 98%, at least 95%, at least 90%, at least 80%, at least70%, or at least 60% sequence identity with the native cargo moiety, aslong as the variant retains or has enhanced biological activity of thenative cargo moiety (e.g., at least about this amount of sequenceidentity to the GenBank Accession Nos. listed in Table 2 and listedabove). In some examples, variant sequences retain substantially thesame amount (or even more) of the native biological function of thecargo moiety, such as the ability to kill or inhibit the growth of atarget cell. A cargo moiety can also be a fragment of the nativesequence that retains a substantial amount of the native biologicalfunction of the protein.

The cargo moieties are engineered to target cells by linking them totargeting moieties. Targeting moieties include agents that can bind tocell surface molecules or targets.

Making Targeted Cargo Proteins

Targeted cargo proteins can be prepared by many routine methods as knownin the art. Targeted cargo proteins, as well as modifications thereto,can be made, for example, by engineering the nucleic acid encoding thetargeted cargo protein using recombinant DNA technology or by peptidesynthesis. Modifications to the targeted cargo protein may be made, forexample, by modifying the targeted cargo protein polypeptide itself,using chemical modifications and/or limited proteolysis. Combinations ofthese methods may also be used to prepare the targeted cargo proteins.

Methods of cloning and expressing proteins are well-known in the art,detailed descriptions of techniques and systems for the expression ofrecombinant proteins can be found, for example, in Current Protocols inProtein Science (Coligan, J. E., et al., Wiley & Sons, New York). Thoseskilled in the art will understand that a wide variety of expressionsystems can be used to provide the recombinant protein. Accordingly, thetargeted cargo proteins can be produced in a prokaryotic host (e.g., E.coli, A. salmonicida or B. subtilis) or in a eukaryotic host (e.g.,Saccharomyces or Pichia; mammalian cells, e.g., COS, NIH 3T3, CHO, BHK,293, or HeLa cells; or insect cells). The targeted cargo proteins can bepurified from the host cells by standard techniques known in the art.

Sequences for various exemplary targeting moieties and cargo moietiesare provided in the Tables 1 and 2. Variants and homologs of thesesequences can be cloned, if an alternative sequence is desired, usingstandard techniques [see, for example, Ausubel et al., Current Protocolsin Molecular Biology, Wiley & Sons, NY (1997 and updates); Sambrook etal., supra]. For example, the nucleic acid sequence can be obtaineddirectly from a suitable organism, such as Aeromonas hydrophila, byextracting mRNA and then synthesizing cDNA from the mRNA template (forexample by RT-PCR) or by PCR-amplifying the gene from genomic DNA.Alternatively, the nucleic acid sequence encoding either the targetingmoiety or the cargo moiety can be obtained from an appropriate cDNAlibrary by standard procedures. The isolated cDNA is then inserted intoa suitable vector, such as a cloning vector or an expression vector.

Mutations (if desired) can be introduced at specific, pre-selectedlocations by in vitro site-directed mutagenesis techniques well-known inthe art. Mutations can be introduced by deletion, insertion,substitution, inversion, or a combination thereof, of one or more of theappropriate nucleotides making up the coding sequence.

The expression vector can further include regulatory elements, such astranscriptional elements, required for efficient transcription of thetargeted cargo protein-encoding sequences. Examples of regulatoryelements that can be incorporated into the vector include, but are notlimited to, promoters, enhancers, terminators, and polyadenylationsignals. Vectors that include a regulatory element operatively linked toa nucleic acid sequence encoding a genetically engineered targeted cargoprotein can be used to produce the targeted cargo protein.

The expression vector may additionally contain heterologous nucleic acidsequences that facilitate the purification of the expressed targetedcargo protein, such as affinity tags such (e.g., metal-affinity tags,histidine tags, avidin/streptavidin encoding sequences,glutathione-S-transferase (GST) encoding sequences, and biotin encodingsequences). In one example, such tags are attached to the N- orC-terminus of a targeted cargo protein, or can be located within thetargeted cargo protein. The tags can be removed from the expressedtargeted cargo protein prior to use according to methods known in theart. Alternatively, the tags can be retained on the targeted cargoprotein, providing that they do not interfere with the ability of thetargeted cargo protein to target and kill (or decrease growth of) cancercells.

As an alternative to a directed approach to introducing mutations intonaturally occurring pore-forming proteins, a cloned gene expressing apore-forming protein can be subjected to random mutagenesis bytechniques known in the art. Subsequent expression and screening of themutant forms of the protein thus generated would allow theidentification and isolation of targeted cargo moieties.

The targeted cargo proteins can also be prepared as fragments or fusionproteins. A fusion protein is one which includes a targeted cargoprotein linked to other amino acid sequences that do not inhibit theability of the targeted cargo protein to selectively target and inhibitcancer cell growth or kill cancer cells. In an alternative example, theother amino acid sequences are short sequences of, for example, up toabout 5, about 6, about 7, about 8, about 9, about 10, about 20, about30, about 50 or about 100 amino acid residues in length. These shortsequences can be linker sequences as described above. Methods for makingfusion proteins are well known to those skilled in the art.

For example U.S. Pat. No. 6,057,133 discloses methods for making fusionmolecules composed of human interleukin-3 (hIL-3) variant or mutantproteins functionally joined to a second colony stimulating factor,cytokine, lymphokine, interleukin, hematopoietic growth factor or IL-3variant. U.S. Pat. No. 6,072,041 to Davis et al. discloses thegeneration of fusion proteins comprising a single chain Fv moleculedirected against a transcytotic receptor covalently linked to atherapeutic protein.

The targeted cargo protein can include one or more linkers, as well asother moieties, as desired. These can include a binding region, such asavidin or an epitope, or a tag such as a polyhistidine tag, which can beuseful for purification and processing of the fusion protein. Inaddition, detectable markers can be attached to the fusion protein, sothat the traffic of the fusion protein through a body or cell can bemonitored conveniently. Such markers include radionuclides, enzymes,fluorophores, chromophores, and the like.

One of ordinary skill in the art will appreciate that the DNA can bealtered in numerous ways without affecting the biological activity ofthe encoded protein. For example, PCR can be used to produce variationsin the DNA sequence which encodes a targeted cargo protein. Suchvariations in the DNA sequence encoding a targeted cargo protein can beused to optimize for codon preference in a host cell used to express theprotein, or may contain other sequence changes that facilitateexpression.

A covalent linkage of a targeting moiety directly to a cargo moiety orvia a linker may take various forms as is known in the art. For example,the covalent linkage may be in the form of a disulfide bond. The DNAencoding one of the components can be engineered to contain a uniquecysteine codon. The second component can be derivatized with asulfhydryl group reactive with the cysteine of the first component.Alternatively, a sulfhydryl group, either by itself or as part of acysteine residue, can be introduced using solid phase polypeptidetechniques. For example, the introduction of sulfhydryl groups intopeptides is described by Hiskey (Peptides 3:137, 1981).

Proteins also can be chemically modified by standard techniques to add asulfhydryl group. For example, Traut's reagent (2-iminothiolane-HCl)(Pierce Chemicals, Rockford, Ill.) can be used to introduce a sulfhydrylgroup on primary amines, such as lysine residues or N-terminal amines. Aprotein or peptide modified with Traut's reagent can then react with aprotein or peptide which has been modified with reagents such asN-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (Pierce Chemicals,Rockford, Ill.).

The components can also be joined using the polymer,monomethoxy-polyethylene glycol (mPEG), as described in Maiti et al.,Int. J. Cancer Suppl., 3:17-22, 1988.

The targeting moiety and the cargo moiety can also be conjugated throughthe use of standard conjugation chemistries as is known in the art, suchas carbodiimide-mediated coupling (for example, DCC, EDC or activatedEDC), and the use of 2-iminothiolane to convert epsilon amino groups tothiols for crosslinking and m-maleimidobenzoyl-n-hydroxysuccinimidylester (MBS) as a crosslinking agent.

Linking of a cargo moiety to a targeting moiety may be direct meaningthat one portion of the cargo moiety is directly attached to a portionof the targeting moiety. For example, one end of the amino acid sequenceof a cargo protein can be directly attached to an end of the amino acidsequence of the targeting moiety. For example, the C-terminus of thecargo protein can be linked to the N-terminus of the targeting moiety,or the C-terminus of the targeting moiety can be linked to theN-terminus of the cargo protein. Methods of generating such fusionproteins are routine in the art, for example using recombinant molecularbiology methods.

In another example, the cargo moiety is linked to the targeting moietyindirectly through a linker. The linker can serve, for example, simplyas a convenient way to link the two entities, as a means to spatiallyseparate the two entities, to provide an additional functionality to thetargeted cargo protein, or a combination thereof.

In general, the linker joining the targeting moiety and the cargo moietycan be designed to (1) allow the two molecules to fold and actindependently of each other, (2) not have a propensity for developing anordered secondary structure which could interfere with the functionaldomains of the two moieties, (3) have minimal hydrophobic or chargedcharacteristic which could interact with the functional protein domainsand/or (4) provide steric separation of the two regions. For example insome instances it may be desirable to spatially separate the targetingmoiety and the cargo moiety to prevent the targeting moiety frominterfering with the inhibitory activity of the targeted cargo moietyand/or the cargo moiety interfering with the targeting activity of thetargeting moiety. The linker can also be used to provide, for example,lability to the connection between the targeting moiety and the cargomoiety, an enzyme cleavage site (for example a cleavage site for aprotease), a stability sequence, a molecular tag, a detectable label, orvarious combinations thereof.

The linker can be bifunctional or polyfunctional, e.g. contains at leastabout a first reactive functionality at, or proximal to, a first end ofthe linker that is capable of bonding to, or being modified to bond to,the targeting moiety and a second reactive functionality at, or proximalto, the opposite end of the linker that is capable of bonding to, orbeing modified to bond to, the cargo moiety being modified. The two ormore reactive functionalities can be the same (i.e. the linker ishomobifunctional) or they can be different (i.e. the linker isheterobifunctional). A variety of bifunctional or polyfunctionalcross-linking agents are known in the art that are suitable for use aslinkers (for example, those commercially available from Pierce ChemicalCo., Rockford, Ill.), such as avidin and biotin. Alternatively, thesereagents can be used to add the linker to the targeting moiety and/orcargo moiety.

The length and composition of the linker can be varied considerablyprovided that it can fulfill its purpose as a molecular bridge. Thelength and composition of the linker are generally selected taking intoconsideration the intended function of the linker, and optionally otherfactors such as ease of synthesis, stability, resistance to certainchemical and/or temperature parameters, and biocompatibility. Forexample, the linker should not significantly interfere with the abilityof the targeting moiety to target the targeted cargo protein to a cancercell, or with the activity of the targeted cargo protein relating toactivation, pore-forming ability, or toxin activity.

Linkers suitable for use may be branched, unbranched, saturated, orunsaturated hydrocarbon chains, as well as peptides as noted above.Furthermore, if the linker is a peptide, the linker can be attached tothe targeting moiety and/or the cargo moiety using recombinant DNAtechnology. Such methods are well-known in the art and details of thistechnology can be found, for example, in Sambrook et al., supra.

In one example, the linker is a branched or unbranched, saturated orunsaturated, hydrocarbon chain having from 1 to 100 carbon atoms,wherein one or more of the carbon atoms is optionally replaced by —O— or—NR— (wherein R is H, or C1 to C6 alkyl), and wherein the chain isoptionally substituted on carbon with one or more substituents selectedfrom the group of (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl,(C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, amide,azido, cyano, nitro, halo, hydroxy, oxo (═O), carboxy, aryl, aryloxy,heteroaryl, and heteroaryloxy. Examples of suitable linkers include, butare not limited to, peptides having a chain length of 1 to 500 aminoacid residues (such as 1 to 100, 1 to 50, 6 to 30, such as less than 30amino acids). Typically surface amino acids in flexible protein regionsinclude Gly, Asn and Ser. Other neutral amino acids, such as Thr andAla, can also be used in the linker sequence. Additional amino acids canbe included in the linker to provide unique restriction sites in thelinker sequence to facilitate construction of the fusions. Otherexemplary linkers include those derived from groups such asethanolamine, ethylene glycol, polyethylene with a chain length of 6 to100 carbon atoms, polyethylene glycol with 3 to 30 repeating units,phenoxyethanol, propanolamide, butylene glycol, butyleneglycolamide,propyl phenyl, and ethyl, propyl, hexyl, steryl, cetyl, and palmitoylalkyl chains.

In one example, the linker is a branched or unbranched, saturated orunsaturated, hydrocarbon chain, having from 1 to 50 carbon atoms,wherein one or more of the carbon atoms is optionally replaced by —O— or—NR— (wherein R is as defined above), and wherein the chain isoptionally substituted on carbon with one or more substituents selectedfrom the group of (C1-C6) alkoxy, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy,(C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, amide, hydroxy, oxo (═O),carboxy, aryl and aryloxy.

In a specific example, the linker is a peptide having a chain length of1 to 50 amino acid residues, such as 1 to 40, 1 to 20, or 5 to 10 aminoacid residues.

Peptide linkers that are susceptible to cleavage by enzymes of thecomplement system, urokinase, tissue plasminogen activator, trypsin,plasmin, or another enzyme having proteolytic activity may be used inone example. According to another example, the targeted cargo proteinincludes a targeting moiety attached via a linker susceptible tocleavage by enzymes having a proteolytic activity such as a urokinase, atissue plasminogen activator, plasmin, thrombin or trypsin. In addition,targeting moieties may be attached to the cargo moiety via disulfidebonds (for example, the disulfide bonds on a cysteine molecule). Sincemany tumors naturally release high levels of glutathione (a reducingagent) this can reduce the disulfide bonds with subsequent release ofthe cargo moiety at the site of delivery.

In one example, the targeted cargo protein includes a targeting moietylinked by a cleavable linker region. In another example, the cleavablelinker region is a protease-cleavable linker, although other linkers,cleavable for example by small molecules, may be used. Examples ofprotease cleavage sites are those cleaved by factor Xa, thrombin andcollagenase. In one example, the protease cleavage site is one that iscleaved by a protease that is associated with a disease. In anotherexample, the protease cleavage site is one that is cleaved by a proteasethat is up-regulated or associated with cancers in general. Examples ofsuch proteases are uPA, the matrix metalloproteinase (MMP) family, thecaspases, elastase, prostate specific antigen (PSA, a serine protease),and the plasminogen activator family, as well as fibroblast activationprotein. In still another example, the cleavage site is cleaved by aprotease secreted by cancer-associated cells. Examples of theseproteases include matrixmetalloproteases, elastase, plasmin, thrombin,and uPA. In another example, the protease cleavage site is one that isup-regulated or associated with a specific cancer. The precise sequencesare available in the art and the skilled person will have no difficultyin selecting a suitable cleavage site. By way of example, the proteasecleavage region targeted by Factor Xa is I E G R. The protease cleavageregion targeted by enterokinase is D D D D K. The protease cleavageregion targeted by thrombin is L V P R G. In one example, the cleavablelinker region is one which is targeted by endocellular proteases.

As known in the art, the attachment of a linker to cargo moiety (or of alinker element to a cleavable element, or a cleavable element to anothercargo moiety) need not be a particular mode of attachment or reaction.

Testing Targeted Cargo Proteins

Targeted cargo proteins can be tested using standard techniques known inthe art. Exemplary methods of testing candidate targeted cargo proteinsare provided below and in the examples included herein. One of ordinaryskill in the art will understand that other methods of testing thetargeted cargo proteins are known in the art and are also suitable fortesting candidate targeted cargo proteins. For example, methods known inthe art for testing for anti-tumor activity can be used. The targetedcargo proteins can initially be screened against a panel of tumor celllines. A cell proliferation assay, such as the WST-1 kit sold by Roche,can be used. Potency can be evaluated using different drugconcentrations in the presence or absence of active agents that inhibitcancer cells. Selected drug candidates from the initial tumor cellscreen can be further characterized through additional in vitro assaysand in relevant xenograft models to examine anti-tumor activity, such asthose described in the Examples herein.

Use of Targeted Cargo Proteins in Combination with Active Agents

Targeted cargo proteins of the invention may be administered byparenteral means, including subcutaneous, intravenous or intramuscularinjection, or by injection into a body cavity. Parenteral administrationby intravenous injection or infusion is preferred. Alternatively,targeted cargo proteins may be administered by direct injection orinfusion into a tumor, for example a brain tumor or a prostate tumor.Alternative methods of administration of the targeted cargo proteinswill be evident to one of ordinary skill in the art. Such methods mayinclude for example, the use of catheters or implantable pumps toprovide continuous infusion over a period of several hours to severaldays into the subject in need of treatment. It is anticipated thatactive agents will be administered by the routes that are currently inuse for their administration in clinical settings.

The dosages of targeted cargo proteins to be administered to a subjectare not subject to absolute limits, but will depend on the nature of thetargeted cargo protein and its unwanted side effects, the subject beingtreated and the type of condition being treated and the manner ofadministration. Generally the dose will be a therapeutically effectiveamount. (A therapeutically effective amount of a targeted cargo proteincan be determined in various ways, such as assaying for improvement ofthe condition of a subject having cancer by monitoring the size of atumor in a subject, the partial or complete alleviation of symptoms,halting the growth of a tumor, or decreasing the size of a tumor.Effective amounts may also be determined through various in vitro or invivo assays similar to the ones described in the Examples providedherein.)

The therapeutically effective dose will also depend on whetheradministration is parenteral or local. For parenteral administration oftargeted cargo proteins, exemplary dosages for administration to asubject for a single treatment may range from 10 ng to 10 mg per squaremeter (m²) of body surface area, from 1 μg to 1 mg per m² of bodysurface area, and from 10 μg to 100 μg per square meter (m²) of bodysurface area. For localized treatment (such as injection or infusioninto a brain tumor) a single treatment may comprise a dosage of targetedcargo protein ranging from 10 ng to 10 mg, from 10 μg to about 1 mg, orfrom 25 μg to 0.5 mg.

Treatments with targeted cargo proteins may be completed in a singleday, or may be done repeatedly on multiple days with the same or adifferent dosage. Repeated treatments may be done on the same day, onsuccessive days, or every 1-3 days, every 3-7 days, every 1-2 weeks,every 2-4 weeks, every 1-2 months, or at even longer intervals.

Dosages of the active agents are determined in accordance with currentclinical protocols for the active agent being used.

It is anticipated that the therapeutic dosages of either the targetedcargo proteins or the active agents when used in combination may bereduced from what would otherwise be determined to be the optimal levelfor each agent administered alone due to the synergy between thetargeted cargo protein and the active agent.

The active agents may be administered concurrently with the targetedcargo proteins, or within hours or days. In some embodiments, the activeagent is administered within 24, 48, 72 or 96 hours of administration ofthe targeted cargo protein. The active agents may be administered moreor less frequently than the targeted cargo proteins. For example, whenrepeat treatments with a targeted cargo protein are given, sometreatments, but not others, may be done in conjunction with an activeagent. Treatment with the active agent may be done more of lessfrequently than treatment with the targeted cargo protein.

Pharmaceutical Compositions

Pharmaceutical compositions can include one or more targeted cargoproteins and/or one or more active agents, and one or more non-toxicpharmaceutically acceptable carriers, diluents, excipients and/oradjuvants. If desired, other active ingredients may be included in thecompositions. As indicated above, such compositions are suitable for usein the treatment of cancer. The term “pharmaceutically acceptablecarrier” refers to a carrier medium which does not interfere with theeffectiveness of the biological activity of the active ingredients andwhich is not toxic to the host or patient. Representative examples areprovided below.

The pharmaceutical compositions may comprise, for example, from about 1%to about 95% of a targeted cargo protein. Compositions formulated foradministration in a single dose form may comprise, for example, about20% to about 90% of the targeted cargo proteins, whereas compositionsthat are not in a single dose form may comprise, for example, from about5% to about 20% of the targeted cargo proteins. Concentration of thetargeted cargo protein in the final formulation can be at least 1 ng/mL,such as at least 1 μg/mL or at least 1 mg/mL. For example, theconcentration in the final formulation can be between about 0.01 μg/mLand about 1,000 μg/mL. In one example, the concentration in the finalformulation is between about 0.01 mg/mL and about 100 mg/mL.

The targeted cargo proteins can be delivered along with apharmaceutically acceptable vehicle. In one example, the vehicle mayenhance the stability and/or delivery properties. Thus, the disclosurealso provides for formulation of the targeted cargo protein with asuitable vehicle, such as an artificial membrane vesicle (including aliposome, noisome, nanosome and the like), microparticle ormicrocapsule, or as a colloidal formulation that comprises apharmaceutically acceptable polymer. The use of such vehicles/polymersmay be beneficial in achieving sustained release of the targeted cargoproteins. Alternatively, or in addition, the targeted cargo proteinformulations can include additives to stabilize the protein in vivo,such as human serum albumin, or other stabilizers for proteintherapeutics known in the art. Targeted cargo protein formulations canalso include one or more viscosity enhancing agents which act to preventbackflow of the formulation when it is administered, for example byinjection or via catheter. Such viscosity enhancing agents include, butare not limited to, biocompatible glycols and sucrose.

Pharmaceutical compositions formulated as aqueous suspensions containthe active compound(s) in admixture with one or more suitableexcipients, for example, with suspending agents, such as sodiumcarboxymethylcellulose, methyl cellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, hydroxypropyl-β-cyclodextrin, gumtragacanth and gum acacia; dispersing or wetting agents such as anaturally-occurring phosphatide, for example, lecithin, or condensationproducts of an alkylene oxide with fatty acids, for example,polyoxyethyene stearate, or condensation products of ethylene oxide withlong chain aliphatic alcohols, for example,hepta-decaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol for example,polyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example, polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives, for exampleethyl, or n-propyl p-hydroxy-benzoate, or one or more coloring agents.

Compositions can be preserved by the addition of an anti-oxidant such asascorbic acid.

The pharmaceutical compositions can be formulated as a dispersiblepowder or granules, which can subsequently be used to prepare an aqueoussuspension by the addition of water. Such dispersible powders orgranules provide the active ingredient in admixture with one or moredispersing or wetting agents, suspending agents and/or preservatives.Suitable dispersing or wetting agents and suspending agents areexemplified by those already mentioned above.

Pharmaceutical compositions can also be formulated as oil-in-wateremulsions. The oil phase can be a vegetable oil, for example, olive oilor arachis oil, or a mineral oil, for example, liquid paraffin, or itmay be a mixture of these oils. Suitable emulsifying agents forinclusion in these compositions include naturally-occurring gums, forexample, gum acacia or gum tragacanth; naturally-occurring phosphatides,for example, soy bean, lecithin; or esters or partial esters derivedfrom fatty acids and hexitol, anhydrides, for example, sorbitanmonoleate, and condensation products of the said partial esters withethylene oxide, for example, polyoxyethylene sorbitan monoleate.

The pharmaceutical compositions containing one or more targeted cargoproteins and/or one or more active agents can be formulated as a sterileinjectable aqueous or oleaginous suspension according to methods knownin the art and using suitable one or more dispersing or wetting agentsand/or suspending agents, such as those mentioned above. The sterileinjectable preparation can be a sterile injectable solution orsuspension in a non-toxic parentally acceptable diluent or solvent, forexample, as a solution in 1,3-butanediol. Acceptable vehicles andsolvents that can be employed include, but are not limited to, water,Ringer's solution, lactated Ringer's solution and isotonic sodiumchloride solution. Other examples include, sterile, fixed oils, whichare conventionally employed as a solvent or suspending medium, and avariety of bland fixed oils including, for example, synthetic mono- ordiglycerides. Fatty acids such as oleic acid can also be used in thepreparation of injectables.

In one example, the targeted cargo protein is conjugated to awater-soluble polymer, e.g., to increase stability or circulating halflife or reduce immunogenicity. Clinically acceptable, water-solublepolymers include, but are not limited to, polyethylene glycol (PEG),polyethylene glycol propionaldehyde, carboxymethylcellulose, dextran,polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polypropyleneglycol homopolymers (PPG), polyoxyethylated polyols (POG) (e.g.,glycerol) and other polyoxyethylated polyols, polyoxyethylated sorbitol,or polyoxyethylated glucose, and other carbohydrate polymers. Methodsfor conjugating polypeptides to water-soluble polymers such as PEG aredescribed, e.g., in U.S. patent Pub. No. 20050106148 and referencescited therein. In one example the polymer is a pH-sensitive polymersdesigned to enhance the release of drugs from the acidic endosomalcompartment to the cytoplasm (see for example, Henry et al.,Biomacromolecules 7(8):2407-14, 2006).

Active agents may be included in a pharmaceutical formulation togetherwith target cargo proteins for co-administration, or may be formulatedseparately. They may be formulated in conventional pharmaceuticallyacceptable carriers. (vehicles) such as those found in Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of active agents or the targetedcargo protein molecules provided herein.

Diseases or Conditions that May be Treated by the Methods of theDisclosure

Diseases or conditions that may be treated using the methods of thedisclosure are characterized by cells that uniquely express, orover-express, at least one target molecule that specifically binds to atargeted cargo protein. Such diseases or conditions may include variousinflammatory conditions as well as benign tumors or malignant tumors(cancer). Tumors can be solid or hematological. Examples ofhematological tumors include, but are not limited to: leukemias,including acute leukemias (such as acute lymphocytic leukemia, acutemyelocytic leukemia, acute myelogenous leukemia and myeloblastic,promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronicleukemias (such as chronic myelogenous leukemia, and chronic lymphocyticleukemia), myelodysplastic syndrome, and myelodysplasia, polycythemiavera, lymphoma, (such as Hodgkin's disease, all forms of non-Hodgkin'slymphoma), multiple myeloma, Waldenstrom's macroglobulinemia, and heavychain disease.

Examples of solid tumors, such as sarcomas and carcinomas, include, butare not limited to: fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, pancreatic cancer, breast cancer, lung cancer, ovariancancer, prostate cancer, benign prostatic hyperplasia, hepatocellularcarcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, Wilms' tumor, epithelial tumors (e.g.,cervical cancer, gastric cancer, skin cancer, head and neck tumors),testicular tumor, bladder carcinoma, melanoma, brain tumors, and CNStumors (such as a glioma, astrocytoma, medulloblastoma,craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, meningioma, neuroblastoma andretinoblastoma).

Preferred diseases and conditions that may be treated by the methods ofthe invention include brain cancer, including malignant astrocytoma andgliobastoma multiforme, Kaposi sarcoma, bladder cancer, renal cellcancer, breast cancer, pancreatic cancer, non-small cell lung cancer,thyroid cancer, squamous cell carcinoma of the head and neck, coloncancer and other cancers of the gastrointestinal system, mesotheliomaand prostate cancer.

EXAMPLES Example 1

We have observed that 42 of 70 (60%) tumor samples from patients withPDA express moderate- to high-density surface IL-4 receptor (IL-4R),whereas normal pancreatic samples express no or low-density IL-4R.PRX321 was specifically and highly cytotoxic [50% protein synthesisinhibition (IC50) ranging from >0.1 to 13 ng/mL] to six of eightpancreatic cancer cell lines, whereas no cytotoxicity (IC50>1,000 ng/mL)was observed in normal human pancreatic duct epithelium cells,fibroblasts, and human umbilical vein endothelial cells (HUVEC). We alsoshowed that PRX321 in combination with gemcitabine exhibited synergisticantitumor activity in vitro. To confirm synergistic antitumor activityin vivo and monitor precise real-time disease progression, we used anovel metastatic and orthotopic mouse model using green fluorescentprotein-transfected cancer cells and whole-body imaging system. Thecombination of both agents caused complete eradication of tumors in 40%of nude mice with small established PDA tumors. In addition, combinedtreatment significantly prolonged the survival of nude mice bearing day14 advanced distant metastatic PDA tumors. Similar results were observedin mice xenografted with PDA obtained from a patient undergoing surgicalresection. These results indicate that PRX321 combined with gemcitabinemay provide effective therapy for the treatment of patients with PDA.

Example 2

In this study, we examined expression of IL-4R in samples derived fromPDA and the efficacy of PRX321, gemcitabine, and combination of both inprimary and metastatic tumor models. To imitate aggressive clinicalsituation and to monitor precise real-time disease progression, we useda novel metastatic and orthotopic advanced pancreatic cancer model usingretroviral green fluorescent protein (GFP)-transfected pancreatic cancercell line and whole-body imaging system (27). Together, our study showsthat PRX321 synergizes with gemcitabine, significantly inhibiting thegrowth of primary and metastatic tumor lesions, prolonging the survivaltime, and completely eradicating tumors in 40% of mice in an earlypancreatic cancer model.

Example 3 Materials and Methods

Cell culture, reagents, and tissue specimens. Cell lines were obtainedfrom the American Type Culture Collection and Sciencell. Humanpancreatic duct epithelium (HPDE) cells were cultured routinely inkeratinocyte serum-free medium supplemented with bovine pituitaryextract and epidermal growth factor (Life Technologies; ref. 28). PRX321[IL4(38-37)-PE38KDEL] was produced as described previously (23). Fifteenparaffin-embedded tissue sections and tissue arrays containing 70 tumorspecimens were obtained from Cooperative Human Tissue Network and U.S.Biomax, respectively. Gemcitabine was procured through the pharmacy ofthe clinical center (NIH).

Immunohistochemistry and flow cytometry. Immunohistochemistry was doneas described previously (24). Deparafinized tissue sections wereincubated with anti-human IL-4Ra polyclonal antibody (Santa CruzBiotechnology) or isotype control (IgG). The results were scored on thebasis of the density of staining 0%, 0% to 10%, 11% to 50%, 51% to 100%as negative, weak, moderate, and strong, respectively. Tissue sectionsfor IL-4R were evaluated by Dr. Satoru Takahashi who is a pathologist atNagoya City University in Japan.

Expression of IL-4Ra on pancreatic cancer cell lines and HPDE cells wasassessed by flow cytometry using phycoerythrin-conjugated anti-IL-4Ramonoclonal antibody as previously described (29). Staining with isotypeMatched IgG served as control. Protein synthesis inhibition assay andassessment of synergism or antagonism. The in vitro cytotoxic activityof PRX321, gemcitabine, and their combination was measured by theinhibition of protein synthesis (18). Drug interaction between PRX321and gemcitabine was assessed at a concentration ratio of 1:1, using thecombination index (CI), where CI<1, CI=1, and CI>1 indicate synergistic,additive, and antagonistic effects, respectively (30). On the basis ofthe isobologram analysis for mutually exclusive effects, the CI valuewas calculated as follows:

${CI} = {\frac{(D)_{1}}{\left( D_{x} \right)_{1}} + \frac{(D)_{2}}{\left( D_{x} \right)_{2}}}$

where (Dx)1 and (Dx)2 are the concentrations of PRX321 and gemcitabine,respectively, required to inhibit cell growth by 50%, and (D)1 and (D)2are the drug concentrations in combination treatments that also inhibitcell growth by 50% (isoeffective compared with the single drugs).Semiquantitative and real-time TaqMan reverse transcription-PCR.Semiquantitative reverse transcription-PCR (RT-PCR) was done asdescribed previously (31). Quantification of IL-4Ra mRNA expressionlevels in pancreatic cancer cell lines was determined by real-timeRT-PCR using a set of IL-4Ra-specific TaqMan probe (5-FAM, 3-MGB) andprimers (Applied Biosystems; ref. 24). Gene expression was normalized toglyceraldehyde-3-phosphate dehydrogenase or h-actin before the foldchange in gene expression was calculated.

Retroviral transduction and selection of high-GFP-expressing MIAPaCa-2pancreatic cancer cells. MIA-PaCa-2 cells expressing GFP wereestablished using a 1:1 precipitated mixture of retroviral supernatantsof the HEK293 cells and RPMI 1640 (Life Technologies, Inc.), asdescribed previously (32).

Animals. Severe combined immunodeficient (SCID) mice and nude nu/nu micebetween age 5 and 6 weeks were maintained in a barrier facility onHEPA-filtered racks. All animal studies were conducted under an approvedprotocol in accordance with the principles and procedures outlined inthe NIH Guideline for the Care and Use of Laboratory Animals.

Whole-body imaging. The tumor-bearing mice were periodically examined ina fluorescence light box illuminated by fiberoptic light at 440/20 nmwavelength (Lightools Research, Inc.). Emitted fluorescence wascollected through a long-pass filter GG475 (Chroma Technology) on aHamamatsu C5810 3-chip cooled color charge coupled device camera(Hamamatsu Photonics Systems). Real-time determination of tumor burdenwas done by quantifying fluorescent surface area as described previously(32).

Surgical orthotopic implantation of MIA-PaCa-2-GFP tumors.MIA-PaCa-2-GFP cells were injected s.c. into the right dorsal flank ofnude mice. Pancreatic tumors, grown s.c. in nude mice, were cut withscissors and minced into f3% 3% 3-mm pieces. For orthotopic surgery, thepancreas was carefully exposed, and tumor chunks were transplanted onthe middle of the pancreas with a 6-0 Dexon surgical suture (Davis-Geck,Inc.). The pancreas was then returned to the peritoneal cavity, theabdominal wall, and the skin was closed with 6-0 Dexon sutures.

Experimental design and treatment. For early pancreatic cancer model,primary tumor lesions were detected by external whole-body imaging onday 4 after transplantation. Once the tumors were visualized, mice wererandomized into four groups of 10 each. Treatment was initiated on day5. For advanced pancreatic cancer model, primary and metastasis tumorlesions were detected by external whole-body imaging on day 14post-transplantation of tumor chunk and randomized into four groups of10 mice each. Treatment was initiated on day 15.

Primary and orthotopic pancreatic cancer model using a clinical sample.Primary pancreatic cancer specimens were obtained from a patientundergoing radical pancreatectomy at National Cancer Institute underinstitutional review board-approved protocol. Viable tumor tissue fromspecimen was cut into small pieces (3% 3% 3 mm) and implanted in thepancreas of 5- to 6-week-old male SCID mice. Primary xenografts werepropagated continuously in SCID mice for in vivo testing. Clinicalsample-bearing mice were also treated after day 31 by the same protocolas described above.

Statistical analysis. The mean tumor volume in therapeutic and controlgroups was analyzed by ANOVA. Survival curves were generated byKaplan-Meier method and compared by using the log-rank test.

Results Expression of IL-4R in PDA Tissues.

Tissue sections from 15 normal pancreas and 70 PDA specimens wereanalyzed by immunohistochemical analysis for the expression of IL-4Ra(data not shown). Tumor specimens showed weak to strong staining forIL-4Ra in PDAs. Only weak staining was observed in tumor-infiltratingstromal fibroblasts and endothelial cells. When the proportion ofIL-4Ra-positive cancer cells was counted, 23 of 70 primary tumorsclassified into strong expression group, 19 into moderate expressiongroup, 11 into weak expression group, and 17 into the negativeexpression group. Thus, 42 of 70 (60%) PDA samples expressed moderate tohigh density IL-4Ra. In contrast, only 2 of 15 normal pancreas samplesshowed weak staining for IL-4Ra in normal acinar and ductal cells.

Pancreatic Cancer Cell Lines Expressing IL-4R are Sensitive to PRX321.

We examined the expression of IL-4Ra mRNA by RT-PCR and real-time RT-PCRin eight pancreatic cancer and one normal HPDE cell lines. Six of eightcancer cell lines showed varied density of IL-4Ra mRNA expression,whereas HPAF-II, PK-1, and HPDE cell lines showed no expression (datanot shown). Real-time RT-PCR analysis confirmed conventional RT-PCRresults and showed that MIA-PaCa-2 and SW1990 cell lines expresseshighest level of IL-4Ra mRNA, followed by Capan-1, ASPC-1, Panc-1, andHS766T cell lines. Flow cytometric analysis confirmed mRNA expressiondata and showed that IL-4Ra is expressed on the cell surface of threepancreatic cancer cell lines but not in normal HPDE cells.

Next, we determined the sensitivity of pancreatic cancer cell lines toPRX321 by protein synthesis inhibition assay, which has been shown to bedirectly proportional to cell death (19). PRX321 inhibited proteinsynthesis of pancreatic cancer cell lines in a concentration-dependentmanner. MIA-PaCa-2 and SW1990 cell lines were extremely sensitive to thecytotoxin (IC50 0.08 and 0.36 ng/mL, respectively), followed by Capan-1(IC50 7 ng/mL) and HS766T (IC50 13 ng/mL;). IC50 in Panc-1 and ASPC-1cell lines was <10 ng/mL. Consistent with the lack of IL-4Ra mRNAexpression, HPAF-II and PK-1 cell lines were not sensitive to PRX321(IC50 z 1,000 ng/mL; data not shown).

The cytotoxic activity of PRX321 was neutralized by incubation with anexcess of IL-4, suggesting specific cytotoxicity through binding ofPRX321 to IL-4R (data not shown). We also examined the cytotoxicity ofPRX321 in fibroblast, HUVEC, and HPDE cell lines, because some of thespecimens revealed weak expression of IL-4Ra in nontumor cells. However,PRX321 was not found to be cytotoxic to these cells (IC50 z 1,000ng/mL;). The PRX321 cytotoxic activity correlated with extent of IL-4Raexpression. For example, MIAPaCa-2 cells showed lowest IC50 and highestdensity IL-4R expression as determined by flow cytometric and real-timePCR analyses whereas PK-1 cell line showed highest IC50 as this cellline showed undetectable level of mRNA expression. We also used anothercytotoxin IL-13 Pseudomonas exotoxin, an IL-13 receptor specific fusionprotein (12), to assess the cytotoxicity to pancreatic cancer cell line.However, IL-13 cytotoxin was not cytotoxic to HPAF-II cells (IC50 z1,000ng/mL).

Synergistic Cytotoxicity of PRX321 and Gemcitabine in Pancreatic CancerCell Lines.

Gemcitabine alone mediated a dose-dependent inhibition of proteinsynthesis with IC50 of 22 nmol/L in MIA-PaCa-2 cells, 3.2 nmol/L inCapan-1 cells, 1,000 nmol/L in SW1990 cells, and 14 nmol/L in HS766Tcells (Table 3). When it was combined with PRX321, the protein synthesisinhibition in MIA-PaCa-2 cells was greatly enhanced: IC50 of PRX321became 0.012, 0.001, and 0.00004 ng/mL by adding 0.03, 0.3, and 3 nmol/Lgemcitabine, respectively. These same phenomena were also observed inSW1990 and Capan-1 cells, but not in HS766T cells. The combination indexat IC50 and IC75 (concentration of drug causing 75% inhibition ofprotein synthesis) in MIA-PaCa-2, SW1990, and Capan-1 cells was <1 atall concentrations of gemcitabine (Table 3).

TABLE 3 Cytotoxicity of IL-4 cytotoxin (PRX321), gemcitabine, and theircombination in pancreatic cancer cell lines Cancer cell line Drug IC50*IC75** MIA- IL-4 cytotoxin 0.065 ng/mL 0.32 ng/mL PaCa-2 Gemcitabine 22nmol/L 280 nmol/L Capan-1 IL-4 cytotoxin 3.5 ng/mL 22 ng/mL Gemcitabine3.2 nmol/L 9 nmol/L SW1990 IL-4 cytotoxin 0.36 ng/mL 1 ng/mL Gemcitabine1,000 nmol/L 3,000 nmol/L CI## IC50 IC75 MIA- IL-4 cytotoxin + 0.1530.563 PaCa-2 gemcitabine 0.03 nmol/L IL-4 cytotoxin + 0.0336 0.094gemcitabine 0.3 nmol/L IL-4 cytotoxin + 0.137 0.0138 gemcitabine 3nmol/L Capan-1 IL-4 cytotoxin + 0.287 0.34 gemcitabine 0.003 nmol/L IL-4cytotoxin + 0.026 0.0713 gemcitabine 0.03 nmol/L IL-4 cytotoxin + 0.0960.0603 gemcitabine 0.3 nmol/L IL-4 cytotoxin + 0.938 0.401 gemcitabine 3nmol/L SW1990 IL-4 cytotoxin + 0.5 0.65 gemcitabine 0.003 nmol/L IL-4cytotoxin + 0.27 0.5 gemcitabine 0.03 nmol/L IL-4 cytotoxin + 0.021 0.3gemcitabine 0.3 nmol/L IL-4 cytotoxin + 0.014 0.19 gemcitabine 3 nmol/LIL-4 cytotoxin + 0.03 0.11 gemcitabine 30 nmol/L IL-4 cytotoxin + 0.30.31 gemcitabine 300 nmol/L NOTE: CI < 1, CI = 1, and CI > 1 indicatesynergistic, additive, and antagonistic effects, respectively. *Fiftypercent protein synthesis inhibition. **Seventy-five percent proteinsynthesis inhibition. ##CI values were calculated using the formuladescribed in Materials and Methods.

Example 4 In Vivo Whole-Body Optical Imaging of PDA

We developed pancreatic cancer models to investigate antitumor effectsof PRX321 and showed its correlation with imaging studies in vivo.Pancreatic cancer cells were transfected with GFP. Our transfectiontechnique using retroviral vector revealed consistent bright GFPfluorescence of MIA-PaCa-2 cells. There was no significant difference inmorphology, growth rate, and sensitivity to PRX321 between parent andGFP-transfected cells (data not shown). GFP-transfected MIA-PaCa-2 tumorchunks were orthotopically transplanted to pancreas of nude mice. Thesetumor pieces were derived from MIA-PaCa-2-GFP cells transplanted s.c.GFP fluorescence enabled real-time and sequential whole-body imaging oftumors. Noninvasive quantitative measurements of external visiblefluorescent area enabled the construction of in vivo tumor growthcurves, which seem to correlate with visible tumor growth (see FIGS. 2Aand 3A).

Example 5 Complete Eradication of Tumors by Combination of PRX321 andGemcitabine in an Early Tumor Model

Small primary tumor lesions on day 4 after transplantation were observedin all mice by the real-time whole-body imaging (average fluorescentarea 26.17 F 4.19 mm2). Treatment was initiated on day 5 aftertransplantation. Group 1 animals (negative control) did not receive anytreatment. Group 2 animals received gemcitabine (150 mg/kg) by i.p.administration twice a week as long as the experiment lasted. Group 3animals received PRX321 (100 ug/kg by i.p. route twice a day for 5 days.Group 4 animals were treated with the combination of gemcitabine andPRX321. The results are shown in FIG. 2A. Imaging studies on days 14 and24 confirmed the significant primary tumor growth and metastasis in thenon-treatment group. In contrast, gemcitabine or PRX321 treatment groupshowed a reduction in the rate of tumor growth, compared withnon-treatment group. The PRX321 treatment group showed no tumor lesionsin 6 of 10 mice on day 14, although tumor recurred by day 34.Remarkably, the combination treatment group revealed significantsuppression of tumor growth of primary tumor lesions. Tumor lesions wereundetectable in all 10 mice on day 14. By day 44, 6 of 10 mice showedlocal recurrence and distant metastasis. The rest of the four miceshowed complete eradication of tumor and mice remained tumor-freethrough day 94 when the experiment was terminated.

Example 6 Synergistic Increase in Survival of Mice Treated with aCombination of PRX321 and Gemcitabine in an Early Tumor Model

Median survival time of the animals treated in Example 5 was 27 days innon-treatment group, whereas it was significantly increased to 54, 64,and 92 days in gemcitabine group (P<0.0001), PRX321 group (P<0.0001),and their combination group (P<0.0001) compared with non-treatmentgroup, respectively. Compared with gemcitabine group, significantprolonged survival time was observed in PRX321 group (P=0.017) and thecombination group (P<0.0001). Increase in significant survival advantagecorrelated with tumor area as detected by GFP fluorescence. Prolongedsurvival time in the combination group was 341% compared with thenon-treatment group. Kaplan-Meier survival curves are shown in FIG. 2B.In addition, we did not observe any organ toxicity in heart, liver,lung, kidney, and spleen of PRX321-injected mice evaluated by histologicexamination.

Example 7 Real-Time Imaging of Tumor Growth of the Primary andMetastasis Lesion in an Advanced In Vivo Model

As approximately 85% patients with PDA are diagnosed at an advancedstage at initial diagnosis, an advanced PDA in vivo model needs to beestablished to imitate the clinical situation and to monitor the diseaseand treatment effect (33). Fluorescence imaging on day 14post-transplantation confirmed the tumor growth of primary lesions inall mice and also detected the metastasis lesions to liver, lymph nodes,and peritoneal locations in 40 of 62 mice. Six mice showed metastaticlesions to liver or lymph nodes around hepatoduodenum ligament, 8 showedmetastasis lesions corresponding to peritoneal locations, and 26 withboth metastasis lesions. We did not include mice with the GFP spot atspleen as a metastasis group. Forty mice with confirmed primary andmetastasis tumor lesions on day 14 post-transplantation were dividedinto four groups and treated as described in Materials and Methods(average fluorescent area 94.67 F 8.31 mm2). Group 1 animals (negativecontrol) did not receive any treatment. Group 2 animals receivedgemcitabine (150 mg/kg) by i.p. administration twice a week as long asthe experiment lasted. Group 3 animals received PRX321 (100 ug/kg byi.p. route twice a day for 5 days. Group 4 animals were treated with thecombination of gemcitabine and PRX321. Treatment was done after theconfirmation of metastasis lesions on day 14. The results are shown inFIG. 3A. The real-time whole-body imaging of tumor growth confirmed thesignificant primary tumor growth and metastatic spread on days 14, 21,and 28 after transplantation of tumor in non-treatment control group.Gemcitabine and PRX321 treatment group showed a reduction in the rate oftumor growth compared with the non-treatment group. Especially, thecombination treatment group revealed significant suppression of tumorgrowth at primary and metastasis tumor lesions. The reduction in tumorsize on day 28 was 39.8% in the gemcitabine group (P<0.001), 71.2% inthe PRX321 group (P<0.001), and 79.6% in the combination group (P<0.001)compared with the no treatment group.

Example 8 Combination of PRX321 and Gemcitabine Prolongs the Survival ofMice with Advanced Orthotopic Pancreatic Tumor

We examined the efficacy of PRX321 on the survival of animals in theadvanced PDA model in Example 7. Median survival time of animals was 28days in non-treatment group, whereas it was significantly increased to34, 43, and 52 days in gemcitabine group (P=0.0089), PRX321 group(P<0.0001), and their combination group (P<0.0001), respectively.Compared with gemcitabine group, significant prolonged survival time wasalso observed in the PRX321 group (P=0.0047) and the combination group(P=0.0002). Prolonged survival time in the combination group was 186%compared with the non-treatment group. Increase in significantprolongation of survival correlated with tumor area as detected bywhole-body imaging. Kaplan Meier survival curves are shown in FIG. 3B.

Example 9 Expression of IL-4R in a Clinical Sample and Development ofOrthotopic Xenograft Tumor Model

We obtained a tumor tissue sample that was surgically resected atSurgery Branch at NIH and pathologically diagnosed as moderatelydifferentiated adenocarcinom. This tumor section showed strong stainingfor IL-4Ra in the ductal adenocarcinoma cells and faint staining offibroblasts. We also established tumor and fibroblast cells culturedfrom this sample to examine the antitumor activity of PRX321. The cancercells expressing IL-4R were highly sensitive to PRX321 (IC50 0.32ng/mL), whereas fibroblast cells were not sensitive (IC50 z1,000ng/mL;).

Example 10 PRX321, Gemcitabine, and their Combination SignificantlyProlonged Survival of Mice Transplanted with a Clinical PancreaticCancer Sample

The clinical sample described in Example 9 was orthotopicallytransplanted on the pancreas of SCID mice and when tumors grew, theywere harvested and then orthotopically propagated in the next set ofSCID mice. All mice showed growth of primary tumor and metastasis tolymph nodes in peritoneum, hepatoduodenum ligament, and para-aorticareas. Seventy-five percent of these mice showed the metastasis lesionto liver when mice were sacrificed 30 days after tumor implantation. Toassess the effect of PRX321 in an advanced metastasis model, a third setof SCID mice were orthotopically implanted with tumor pieces obtainedfrom the second set of mice. These mice, when advanced diseasedeveloped, were divided into four groups on day 31 and treated asdescribed in Materials and Methods. Group 1 animals (negative control)did not receive any treatment. Group 2 animals received gemcitabine (150mg/kg) by i.p. administration twice a week as long as the experimentlasted. Group 3 animals received PRX321 (100 ug/kg by i.p. route twice aday for 5 days (days 31-35). Group 4 animals were treated with thecombination of gemcitabine and PRX321. Median survival time of animalswas 62 days in the non-treatment group, whereas it was significantlyincreased to 86, 102, and 134 days in the gemcitabine group (P=0.0081),PRX321 group (P=0.0006), and combination group (P<0.0001), respectively.Compared with gemcitabine, significant prolonged survival time wasobserved of PRX321-treated mice (P=0.0037) and the combination group(P<0.0001). Prolonged survival time in the combination group was 216%compared with the non-treatment group. Kaplan-Meier survival curves areshown in FIG. 4.

Example 11 Conclusions

These studies support our observations of gemcitabine synergizing withPRX321. Despite synergistic effect with gemcitabine, few combinationshave shown clinical advantage (4-7). For example, although EGFRinhibitor showed synergistic antitumor effect in preclinical models, thesurvival benefits for patients with advanced pancreatic cancer seem verymodest at best. It was later found that mutations in the EGFR gene,which correlate with clinical response, are found in <5% of pancreaticcancer patients (8, 41). Therefore, new effective therapies that do notdepend on receptor mutation are needed. As our results show the survivalbenefit by PRX321 when combined with gemcitabine in both early andadvanced pancreatic cancer models, it is possible that this novelapproach will afford better tumor responses than previously observed.The precise mechanism of synergistic effect of gemcitabine with PRX321is not known. Gemcitabine is a synthetic pyrimidine antimetabolitestructurally related to cytarabine (42). Gemcitabine inhibits DNAsynthesis through inhibition of ribonucleotide reductase and depletionof deoxynucleotide pools. On the other hand, PRX321 inhibits proteinsynthesis after internalization into an endosome. In addition, we havepreviously shown that PRX321 can cause apoptotic cell death of cancercells regardless of the cell cycle status (43). It is possible thatgemcitabine enhances apoptotic cell death induced by PRX321. Becauseapoptosis is a prominent mechanism of cancer cell death, the combinationtherapy of these drugs, which act through different mechanism, may be abeneficial treatment option for patients with PDA.

We studied two types of advanced pancreatic cancer models to show theanti-tumor activity of PRX321 and gemcitabine. In orthotopic model, thefreshly resected clinical tumor was implanted to pancreas of SCID mice.It has been reported that this model recapitulates the natural historyof the clinical disease, including the invasive and metastatic pattern(44). Accordingly, the peritoneal organs, lymph nodes, liver, and spleenof mice in our model showed tumor metastasis and invasion 1 month aftertransplantation. PRX321 and gemcitabine showed remarkable antitumoreffects in this model. In future studies, it will be of interest todetermine whether metastatic lesions to various organs express IL-4R,and after treatment with PRX321 these receptor levels decrease alongwith disappearing tumor. In other orthotopic tumor model, tumor piecesdeveloped from MIPaCa-2-GFP cells by s.c. is implanted to pancreas ofnude mice. In this model, PRX321 as well as gemcitabine caused profoundantitumor effects. These data are compatible with our previous reportthat showed the survival benefit by PRX321 alone in orthotopic early andadvanced animal models using Panc-1 and BxPC-3 pancreatic cancer celllines (19). Although we did not test IL-4R-negative tumor in vivomodels, our previous studies have shown that non-small cell lung cancercell line expressing no or low IL-4R are not sensitive to PRX321 in vivo(20). Similar conclusions were drawn in squamous cell carcinoma of headand neck tumor models (45). Thus, PRX321 and gemcitabine show bettersurvival benefit compared with either agent alone in two pancreatictumor models, one derived from clinical sample and the other derivedfrom MIPaCa-2 cell line.

The whole-body imaging of host visualizes the real-time tumor growth atthe primary site and tumor development at metastasis sites without theinvasive procedures, surgery, anesthesia, or use of contrast medium. Dueto the fact that whole-body imaging has the potential of highcorrelation with MRI in quantifying tumor volume, the precise evaluationof tumor growth rate, metastatic situation, and effectiveness of drugscould all be monitored without sacrificing animals (32, 46). Inaddition, imaging may identify biomarker of tumor response inpreclinical models that can be validated in the clinical trial (47). Arecent article reported that red fluorescent protein showed brighter andless background image compared with GFP, when animals were imaged (48).In our study, we used GFP-transfected cells. Therefore, it is possiblethat we were not able to detect micrometastasis lesions. Nevertheless,we could show that mice developed spontaneous tumor metastasis withinthe short time after orthotopic transplantation, which correlated withshort survival time. In addition, our model showed that PRX321 reducedthe rate of tumor growth, including primary and metastasis lesions for15 and 9 days after treatment in early and advanced model, respectively.

Although PRX321 mediated remarkable antitumor effects in vivo, novisible signs of toxicity and features such as weight loss andinactivity were observed in mice receiving optimal doses of PRX321and/or gemcitabine (data not shown). These results are compatible withprevious studies related to both agents (data not shown; refs. 19, 24,49). Previous studies have shown that low density IL-4R are expressed onnormal immunologic and nonhematopoietic cells (22). Consequently, PRX321is not cytotoxic to these cells. Preclinical toxicity studies in micehave shown that PRX321 is well tolerated up to 475 Ag/kg dose given i.v.(50). As human IL-4 does not bind murine IL-4R, PRX321 has also beenadministered to cynomolgus monkeys, whose IL-4R binds human IL-4. Inthese animals, PRX321 was reasonably tolerated up to a dose of 200 Ag/kggiven i.v. every alternate day for three injections (21). In a phase 1clinical trial, reversible elevation of liver enzymes and injection siteinflammatory reactions were reported after i.v. administration of PRX321at 0.027 mg/m2 (25). As our study shows synergistic effects when PRX321is combined with gemcitabine against pancreatic cancer in vitro and invivo, lower doses of PRX321 may be effective for the treatment ofpatients with PDA when combined with gemcitabine.

In conclusion, these studies provide a novel approach for monitoringtumor response by whole-body imaging of the host. Further studies shouldbe done to evaluate the safety, tolerability, and efficacy of PRX321when combined with gemcitabine in various pancreatic cancer models. Inaddition, because of their synergistic effect, PRX321 in combinationwith gemcitabine should be tested in patients with PDA.

Example 12 Repetitive Therapy of Orthotopic Human Pancreatic Cancer byof IL4-PE

Immunodeficient nude mice were transplanted with Green FluorescenceProtein (GFP) transfected human pancreatic tumor cells (Hs766T)orthotropically on pancreas. Five days after tumor implantation, micewere treated with IL4-PE 100 ug/kg/day, i.p. for one week or everyalternate week for 3 weeks. Tumor size was measured by imaging ofvisible fluorescence area in live animals.

One week administration of IL-4-PE significantly decreased the tumorvolume compared to control. However, repetitive therapy with IL4-PEcaused dramatic regression of established pancreatic tumor growth. Wedid not observe any visible side effects with repetitive therapy ofIL4-PE.

REFERENCES

-   1. Sener S F, Fremgen A, Menck H R, Winchester D P. J Am Coll Surg    1999; 189:1-7.-   2. Tempero M, Plunkett W, Ruiz Van Haperen V, et al. J Clin Oncol    2003; 21:3402-8.-   3. Burris H A, Moore M J, Andersen J, et al. J Clin Oncol 1997;    15:2403-13.-   4. Bruns C J, Solorzano C C, Harbison M T, et al. Cancer Res 2000;    60:2926-35.-   5. Yokoi K, Sasaki T, Bucana C D, et al. Cancer Res 2005;    65:10371-80.-   6. Bergers G, Song S, Meyer-Morse N, Bergsland E, Hanahan D. J Clin    Invest 2003; 111:1287-95.-   7. Fujioka S, Sclabas G M, Schmidt C, et al. Oncogene 2003;    22:1365-70.-   8. Moore M J, Goldstein J, Hamm A, et al. J Clin Oncol. 2005 ASCO    Annual Meeting Proceedings. Vol 23, No. 16S, Part I of II (June 1    Supplement); 2005:1.-   9. Pastan I, Hassan R, Fitzgerald D J, Kreitman R J. Nat Rev Cancer    2006; 6:559-65.-   10. Rand R W, Kreitman R J, Patronas N, Varricchio F, Pastan I, Puri    R K. Clin Cancer Res 2000; 6:2157-65.-   11. Weber F, Asher A, Bucholz R, et al. J Neurooncol 2003;    64:125-37.-   12. Kioi M, Husain S R, Croteau D, Kunwar S, Puri R K. Technol    Cancer Res Treat 2006; 5:239-50.-   13. Kreitman R J, Squires D R, Stetler-Stevenson M, et al. J Clin    Oncol 2005; 23:6719-29.-   14. Nelms K, Keegan A D, Zamorano J, Ryan J J, Paul W E. Annu Rev    Immunol 1999; 17:701-38.-   15. Toi M, Bicknell R, Harris A L. Cancer Res 1992; 52:275-9.-   16. Topp M S, Papadimitriou C A, Eitelbach F, et al., Cancer Res    1995; 55:2173-6.-   17. Stadler W M, Rybak M E, Vogelzang N J. Cancer 1995; 76:1629-33.-   18. Husain S R, Kreitman R J, Pastan I, Puri R K. Nat Med 1999;    5:817-22.-   19. Kawakami K, Kawakami M, Husain S R, Puri R K. Cancer Res 2002;    62:3575-80.-   20. Kawakami M, Kawakami K, Stepensky V A, et al. Clin Cancer Res    2002; 8:3503-11.-   21. Kawakami M, Kawakami K, Puri R K. J Neurooncol 2003; 65:15-25.-   22. Kawakami K, Kawakami M, Puri R K. Crit Rev Immunol 2001;    21:299-310.-   23. Kreitman R J, Puri R K, Pastan I. Proc Natl Acad Sci USA 1994;    91:6889-93.-   24. Kioi M, Takahashi S, Kawakami M, Kawakami K, Kreitman R J, Puri    R K. Cancer Res 2005; 65:8388-96.-   25. Garland L, Gitlitz B, Ebbinghaus S, et al. J Immunother 2005;    28:376-81.-   26. Rainov N G, Heidecke V. J Neurooncol 2004; 66:197-201.-   27. Hoffman R M. Nat Rev Cancer 2005; 10:796-806.-   28. Furukawa T, Duguid W P, Rosenberg L, Viallet J, Galloway D A,    Tsao M S. 16. Am J Pathol 1996; 148:1763-70.-   29. Puri R K, Leland P, Kreitman R J, Pastan I. Int J Cancer 1994;    58:574-81.-   30. Chou T C, Motzer R, Tong Y, Bosl G. J Natl Cancer Inst 1994;    86:1517-24.-   31. Murata T, Obiri N I, Debinski W, Puri R K. Biochem Biophys Res    Commun 1997; 238:90-4.-   32. Bouvet M, Wang J, Nardin S R, et al. Cancer Res 2002;    62:1534-40.-   33. Abbruzzese J L. Semin Oncol 2002; 29:2-8.-   34. Symon Z, Davis M, McGinn C J, Zalupski M M, Lawrence T S. Int J    Radiat Oncol Biol Phys 2002; 53:140-5.-   35. Bocci G, Fioravanti A, Orlandi P, et al. Br J Cancer 2005;    93:319-30.-   36. Pratesi G, Petrangolini G, Tortoreto M, et al. Cancer Res 2005;    65:6388-93.-   37. Chun P Y, Feng F Y, Scheurer A M, et al. Cancer Res 2006;    66:981-8.-   38. O'Connor R, Liu C, Ferris C A, et al. Blood 1995; 86:4286-94.-   39. Kim C N, Bhalla K, Kreitman R J, et al. Leuk Res 1999;    23:527-38.-   40. Polito L, Bolognesi A, Tazzari P L, et al. Leukemia 2004;    18:1215-22.-   41. Gilbert J A, Lloyd R V, Ames M M. N Engl J Med 2005; 353:209-10.-   42. Morgan A. Highlights Oncol Pract 1996; 14:74-9.-   43. Husain S R, Kawakami K, Kawakami M, Puri R K. Mol Cancer Ther    2003; 2: 245-54.-   44. Manzotti C, Audisio R A, Pratesi G. Clin Exp Metastasis 1993;    11:5-14.-   45. Strome S E, Kawakami K, Alejandro D, et al. Clin Cancer Res    2002; 8:281-6.-   46. Bouvet M, Spernyak J, Katz M H, et al. Cancer Res 2005;    65:9829-33.-   47. Saur D, Seidler B, Schneider G, et al. Gastroenterology 2005;    129:1237-50.-   48. Katz M H, Takimoto S, Spivack D, Moossa A R, Hoffman R M,    Bouvet M. J Surg Res 2003; 113:151-60.-   49. Zhang X, Galardi E, Duquette M, Lawler J, Parangi S. Clin Cancer    Res 2005; 11:5622-30.-   50. Puri R K, Hoon D S, Leland P, et al. Cancer Res 1996; 56:5631-7.

1-35. (canceled)
 36. A method of inhibiting a cancer cell thatoverexpresses an IL-4 receptor, comprising: a. contacting said cancercell with a targeted cargo protein comprising a targeting moiety thatspecifically binds to an IL-4 receptor, and a cargo moiety that inhibitsaid cancer cell, and b. contacting said cancer cell with an activeagent.
 37. The method of claim 36, wherein the targeted cargo protein isselected from the group consisting of IL4-BAD and IL-13-BAD, andvariants thereof.
 38. The method of claim 36, wherein the cargo moietyis selected from pro-apoptotic members of the Bcl-2 family of proteinswhich include but are not limited to BAX, BAD, BAT, BAK, BIK, BID BIM,BMF, and BOK, and variants thereof.
 39. The method of claim 36, whereinthe cargo moiety is selected from non-Bcl-2 family of pro-apoptoticproteins which include but are not limited to granzymes and caspases andvariants thereof.
 40. The method of claim 36, wherein said cancer cellis a cell of a cancer selected from the group consisting of braincancer, a hematological cancer, Kaposi sarcoma, bladder cancer, renalcell cancer, breast cancer, pancreatic cancer, non-small cell lungcancer, thyroid cancer, squamous cell carcinoma of the head and neck,colon cancer, bile duct carcinoma, ovarian cancer, a cancer of thegastrointestinal system, mesothelioma, rhabdomyosarcoma, and prostatecancer.
 41. The method of claim 36, wherein said cancer cell is selectedfrom the group consisting of a malignant astrocytoma cell, amedulloblastoma cell, a meningioma cell, and a glioma cell.
 42. Themethod of claim 36, wherein said active agent is selected from the groupconsisting of gemcitabine, doxorubicin, FOLFOX (folinic acid,fluorouracil, and oxaliplatin), premetrexed, irinotecan, temozolamide,cisplatin, oxaliplatin, erlotinib, imatinib, cetuximab, bevacizumab,rituximab, an antibody, or other active agent used to treat cancer.